Eur. J. Immunol. 2023;53:2249925 DOI: 10.1002/eji.202249925 Clausen BE et al. 1 of 96
HIGHLIGHTS
Guidelines for mouse and human DC functional assays
Björn E. Clausen#1,2 , Lukas Amon3, Ronald A. Backer1,2, Luciana Berod#1,4, Tobias Bopp1,5,
Anna Brand2, Sven Burgdorf#6, Luxia Chen7, Meihong Da7, Ute Distler#1,5, Regine J. Dress8,
Diana Dudziak#3,9,10, Charles-Antoine Dutertre11,12, Christina Eich2, Anna Gabele1,5,
Melanie Geiger13,14, Florent Ginhoux#11,12,15,16,17, Lucila Giusiano18, Gloria J. Godoy18,
Ahmed E.I. Hamouda13,14, Lukas Hatscher3, Lukas Heger3, Gordon F. Heidkamp3,
Lola C. Hernandez19, Lukas Jacobi3, Tomasz Kaszubowski3, Wan Ting Kong11,12,
Christian H. K. Lehmann3,9,10 , Tamara López-López19, Karsten Mahnke#7, Dominik Nitsche6,
Jörg Renkawitz#20, Rifat A. Reza20, Pablo J. Sáez#19, Laura Schlautmann6, Madeleine T. Schmitt20,
Anna Seichter3, Malte Sielaff1,5, Tim Sparwasser1,18, Patrizia Stoitzner21, Giorgi Tchitashvili3,
Stefan Tenzer#1,5,22, Nounagnon R. Tochoedo3, Damir Vurnek3, Fabian Zink6
and Thomas Hieronymus#13,14,23
1 Research Center for Immunotherapy (FZI), University Medical Center of the Johannes-Gutenberg University Mainz, Mainz,
Germany
2 Institute for Molecular Medicine, Paul Klein Center for Immune Intervention, University Medical Center of the Johannes
Gutenberg-University Mainz, Mainz, Germany
3 Laboratory of Dendritic Cell Biology, Department of Dermatology, University Hospital Erlangen, Germany
4 Institute of Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Germany
5 Institute of Immunology, Paul Klein Center for Immune Intervention, University Medical Center of the Johannes-Gutenberg
University Mainz, Mainz, Germany
6 Laboratory of Cellular Immunology, LIMES Institute, University of Bonn, Bonn, Germany
7 Department of Dermatology, University Hospital Heidelberg, Heidelberg, Germany
8 Institute of Systems Immunology, Hamburg Center for Translational Immunology (HCTI), University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
9 Medical Immunology Campus Erlangen (MICE), Erlangen, Germany
10 Deutsches Zentrum Immuntherapie (DZI), Germany
11 Gustave Roussy Cancer Campus, Villejuif, France
12 Institut National de la Santé et de la Recherche Médicale (INSERM) U1015, Equipe Labellisée—Ligue Nationale contre le
Cancer, Villejuif, France
13 Institute for Biomedical Engineering, Department of Cell Biology, RWTH Aachen University, Medical Faculty, Aachen,
Germany
14 Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany
15 Singapore Immunology Network (SIgN), Agency for Science, Technology and Research, Singapore, Singapore
16 Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of
Medicine, Shanghai, China
17 Translational Immunology Institute, SingHealth Duke-NUS Academic Medical Centre, Singapore, Singapore
18 Institute of Medical Microbiology and Hygiene, University Medical Center of the Johannes Gutenberg-University Mainz,
Germany
19 Cell Communication and Migration Laboratory, Institute of Biochemistry and Molecular Cell Biology, Center for
Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Correspondence: Prof. Björn E. Clausen and Dr. Thomas Hieronymus #Section lead authors
e-mail: bclausen@uni-mainz.de; Thomas.Hieronymus@rwth-aachen.de
© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH www.eji-journal.eu
This is an open access article under the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in anymedium,provided the original work is properly cited.
2 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
20 Biomedical Center (BMC), Walter Brendel Center of Experimental Medicine, Institute of Cardiovascular Physiology and
Pathophysiology, Klinikum der Universität, LMU Munich, Munich, Germany
21 Department of Dermatology, Venerology & Allergology, Medical University Innsbruck, Innsbruck, Austria
22 Helmholtz Institute for Translational Oncology Mainz (HI-TRON Mainz), Mainz, Germany
23 Institute of Cell and Tumor Biology, RWTH Aachen University, Medical Faculty, Germany
This article is part of the Dendritic Cell Guidelines article series, which provides a collec-
tion of state-of-the-art protocols for the preparation, phenotype analysis by flow cytome-
try, generation, fluorescence microscopy, and functional characterization of mouse and
human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues.
Recent studies have provided evidence for an increasing number of phenotypically distinct
conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but
on the other hand are characterized by their tissue- and context-dependent functional
specialization. Here, we describe a selection of assays for the functional characterization
of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis
andmetabolism, followed by guidelines for transcriptomic and proteomic characterization
of cDC populations. Then, a larger group of assays describes the characterization of cDC
migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome
and antigen (cross)-presentation activity. While all protocols were written by experienced
scientists who routinely use them in their work, this article was also peer-reviewed by
leading experts and approved by all co-authors, making it an essential resource for basic
and clinical DC immunologists.
Keywords: dendritic cell  endocytosis  langerhans cell  migration  T cell response
Contents
1 Direct analysis of internalization of proteins (including antibodies) into mouse DC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.1 Preparation of stocks and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.2 Preparation of protein or antibody solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.3 Coupling of the DIBO-SE linker to the protein or antibody of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3.4 Click chemistry reaction of DIBO-SE coupled antibody or protein with the Atto647N-SIP-N3 oligo . . . . . . . . . . . . . . 9
1.3.5 Titration of the labeled antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.6 Titration of the labeled BBQ650-quencher . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3.7 Internalization assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2 Mitochondrial function assessment of mouse Fms-like tyrosine kinase 3 ligand derived-DC using an extracellular flux
analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.1 Generation of FLT3L-DC (FL-DC) cultures from mouse bone marrow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
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Eur. J. Immunol. 2023;53:2249925 GUIDELINES FOR MOUSE AND HUMAN DC FUNCTIONAL ASSAYS 3 of 96
2.3.2 Seahorse cell mito stress assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.3 Normalization of Agilent Seahorse XF data by nuclear cell counting using a BioTek Cytation 5 . . . . . . . . . . . . . . . . . . 16
2.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3 Single cell RNA sequencing of human tissue DC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.1 Preparation of stocks and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.2 Index-sorting of human DC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.3 Generation of SMARTseq2 single-cell transcriptome data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.4.1 Pre-processing, quality assessment and control, and analysis of SMARTseq2 single-cell transcriptome data . . . . . 21
3.4.2 Dimensionality reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.4.3 Defining clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.4.4 Plotting the expression of genes or proteins of interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.4.5 Integrating indexed-flow cytometry data with scRNAseq data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4 FACS isolation and mass spectrometry-based proteomic analysis of rare mouse DC populations . . . . . . . . . . . . . . . . . . . . . . . 24
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.3.1 Single-cell preparation for FACS analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.3.2 Flow cytometric purification, collection and lysis of splenic cDC subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.3.3 Proteolytic digestion using single-pot solid-phase-enhanced sample preparation (SP3) . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.3.4 Liquid chromatography-mass spectrometry (LC-MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.7 Summary table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5 Gelatin degradation assay to address podosome formation and function of mouse DC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.3.1 Coating of coverslips with fluorescently labeled gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.3.2 Preparation and seeding of DC and performing the gelatin degradation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5.3.3 Processing cells for podosome detection and fluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5.3.4 Imaging of podosome formation and activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
5.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6 The under-agarose migration assay: a simple method to visualize and quantify mouse DC migration in confined 3D-like
microenvironments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
 15214141, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/eji.202249925 by Universitätsbibliothek Mainz, Wiley Online Library on [07/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
4 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
6.3.1 Preparation of the ‘under-agarose’ assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
6.3.2 Injection of DC into the ‘under-agarose’ assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
6.3.3 Live-cell Imaging of DC in the ‘under-agarose’ assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
6.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
6.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
7 Quantification of DC migration in 3D collagen matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
7.3.1 Generation of bone marrow derived mouse dendritic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
7.3.2 Preparation of collagen gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
7.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
7.4.1 Analysis of DC migration in collagen (3D environments) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
7.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
8 Analyzing mouse skin DC migration by whole skin explant culture and FITC painting assays . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.2.1 General reagents required for the crawl-out and FITC painting assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.2.2 General solutions for the crawl-out and FITC painting assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.2.3 Reagents specific for the crawl-out assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8.2.4 Solutions specific for the crawl-out assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.2.5 Reagents specific for the FITC painting assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.2.6 Solutions specific for the FITC painting assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.2.7 General equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.2.8 Equipment specific for the crawl-out assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.2.9 Equipment specific for the FITC painting assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.3 Step-by-step sample preparation for the crawl-out assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.3.1 Preparation of ear tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.3.2 Harvesting cells after 24h and 48h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.4 Step-by-step sample preparation for the FITC painting assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.4.1 Applying FITC reagent mix on mouse ear skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.4.2 Single-cell suspension of lymph nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
8.5 General flow cytometry staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
8.6 Data analysis & gating strategy of migrated ear DC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
8.7 Pitfalls and top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
9 Measuring inflammasome activity in human primary DC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
9.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
9.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
9.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
9.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
9.3.1 Preparation of stocks and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
9.3.2 Stimulation of the NLRP3 inflammasome in human DC with ATP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
9.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
9.4.1 Measuring of secreted IL-1β . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
9.4.2 Measuring of inflammasome-induced pyroptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
9.4.3 Measuring of active caspase-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
9.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
9.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
9.7 Summary table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
10 In vitro antigen-presentation assays with murine DC and CD4+ T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
 15214141, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/eji.202249925 by Universitätsbibliothek Mainz, Wiley Online Library on [07/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Eur. J. Immunol. 2023;53:2249925 GUIDELINES FOR MOUSE AND HUMAN DC FUNCTIONAL ASSAYS 5 of 96
10.1.1 DC as antigen presenting cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
10.1.2 The choice of responder T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
10.1.3 The choice of viable or fixed DC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
10.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
10.2.1 Chemicals and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
10.2.2 Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
10.2.3 Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
10.2.4 Buffers and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
10.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
10.3.1 Preparation of single-cell suspensions from lymph nodes and spleen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
10.3.2 Isolation of CD4+ T cells from single-cell suspensions of spleens and lymph nodes (CD4+ T cells Isolation Kit,
Miltenyi Biotec1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
10.3.3 Labeling of Cell Proliferation Dye eFluorTM 450 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
10.3.4 Setting up T Cell proliferation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
10.3.5 Staining of CD4+ T cells and analysis by flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
10.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
11 Quantitative analysis of in vitro cross-presentation by mouse DC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
11.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
11.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
11.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
11.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
11.3.1 Preparation of stocks and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
11.3.2 Generation of bone marrow-derived DC (BM-DC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
11.3.3 Quantification of OVA cross-presentation by flow cytometry using the 25-D1.16 antibody . . . . . . . . . . . . . . . . . . . . . . 70
11.3.4 Quantification of cross-presentation after co-culture with antigen-specific T cells by ELISA . . . . . . . . . . . . . . . . . . . . . 70
11.3.5 Monitoring proliferation of activated T cells by flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
11.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
11.4.1 Analysis of cross-presentation using the 25-D1.16 antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
11.4.2 Analysis of OT-I T cell activation by ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
11.4.3 Analysis of OT-I T cell proliferation by flow cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
11.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
11.5.1 Use of inhibitors/small molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
11.5.2 Sensitivity of the 25-D1.16 antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
11.5.3 Calculation of the division index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
11.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
12 In vivo analysis of killing efficacy by CD8+ T cell responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
12.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
12.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
12.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
12.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
12.3.1 Preparation of stocks and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
12.3.2 Immunization of mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
12.3.3 Preparation of splenocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
12.3.4 CFSE and CTV labeling of target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
12.3.5 Peptide loading of target cells and injection into the immunized mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
12.3.6 Analysis of killing efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
12.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
12.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
12.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13 Measuring naïve T cell responses induced by human primary DC .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13.2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13.2.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
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13.2.2 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13.3 Step-by-step sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13.3.1 Preparation of stocks and solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
13.3.2 Measuring naïve T cell responses using mixed leukocyte reactions with primary DC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
13.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
13.4.1 Analysis of naïve CD4+ T cell proliferation, activation, and polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
13.4.2 Analysis of naïve CD8+ T cell proliferation, activation, and phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
13.5 Pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
13.6 Top tricks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
13.7 Summary table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Author contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Data availability statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
1 Direct analysis of internalization of become fluorescent. However, the here presented method to mea-
proteins (including antibodies) into mouse sure endocytosis does solely focus on internalization and does
DC not require any other step (such as acidification or proteolytic
cleavage).
1.1 Introduction With the here presented method, it is not only possible
to observe multiple populations at the same time (e.g., in
Dendritic cells (DC) belong to the most potent regulators of the total splenocytes, in which different cell types may uptake
adaptive immune response. This includes the induction of new the protein or antibody of interest), but also to directly
and re-call responses to pathogens, the resolution of inflamma- asses endocytosis and observe this DC function over a longer
tion, and the dampening of immune responses to harmless self- period. Especially the longer observation period is needed, if
antigens [1]. A very important prerequisite for the sentinel func- it is planned to study receptor-mediated endocytosis medi-
tion of DC is their ability to uptake material from their surround- ated by fast recycling receptors, such as Fc receptors [3,
ings. Therefore, DC can utilize phagocytosis, pinocytosis as well 7].
as receptor-mediated endocytosis. Many methods have been pro- To directly measure endocytosis, a special labeling of the pro-
posed allowing to study the uptake of material by DC. These tein or antibody of interest is used (see Fig. 1). Therefore, an Oligo
include fluorescently labeled particles and proteins. To specifically coupled to a fluorescent molecule will be coupled to the protein or
assess receptor-mediated endocytosis into DC, different assays antibody to be investigated. After incubation of the cells of inter-
have been developed. These include the indirect assessment of est, such as pure or enriched DC populations or splenic single-cell
endocytosis of antibodies and proteins by staining of the pro- suspensions, the fluorescence of the proteins or antibodies still
teins staying outside after a certain incubation time [2–4] and the bound outside of the cells, will be quenched by using a comple-
removal of proteins bound to the outside of the cells, e.g., by acid mentary Oligo labeled with a compatible quencher molecule. This
wash [5, 6]. Both assay formats have major drawbacks: 1) an indi- method was adapted from Liu et al., Reuter et al., and Lehmann
rect assay does not allow for the distinction of receptor-mediated et al. [3, 5, 8].
endocytosis from the receptor shedding or loss of protein due to This assay works best with freshly isolated, immature DC as
a low affinity, while one can also not observe endocytosis over a maturation often lowers their endocytic capacity. Therefore, it is
longer period of time; 2) while the acid wash allows for detec- very important to ensure that no endotoxin is introduced in this
tion of molecules enriching inside the cell, the acid wash itself, assay. Therefore, the reagents have to be tested for endotoxin
even being only a mild pH change, compromises often many dif- contamination. We also recommend using the DC (or cell suspen-
ferent surface molecules and therefore the different cell subsets sions) directly in this assay to minimize culture time as this also
might not be clearly identified [5]. An endocytosis assay utilizing might affect their maturation status and functional capacity. In
directly fluorescent particles or proteins suffers from similar lim- many cases, DC are also more stable within a complete cell sus-
itations as it is not possible to distinguish just bound particles or pension from an organ, such as spleen, so we recommend to avoid
proteins from the endocytosed. Alternative, also very useful meth- cell enrichment, if possible. In principle, any source for DC from
ods, involve the usage of fluorescent dyes or conjugates which any tissue can be used, but it is important to ensure the isolation
need a pH change (e.g., phRodo) or a proteolytic degradation to in an immature state.
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Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 Direct analysis of internalization of proteins (including antibodies) into mouse DC 7 of 96
Figure 1. Graphical representation of the method. A) Graphical visualization for the coupling of the antibody (or protein) used in the endocytosis
assay. B) Graphical visualization for the endocytosis assay. Cellular images are provided and adapted from Servier Medical Art (smart.servier.com)
and these images are licensed under the Creative Commons Attribution 3.0 Unported License (creativecommons.org/licenses/by/3.0/).
1.2 Materials 1.2.2 Equipment
1.2.1 Reagents Necessary equipment is listed in Table 2.
All reagents are listed in Table 1.
Table 1. Reagents, chemicals, and solutions
Reagent Manufacturer Ordering
number
Chemicals & Solutions
Pure antibody or protein of interest
(important: no azid ions, no glycin, no glycerine, no stabilizer proteins –
buffer exchange possible)
Dulbecco´s Phosphate Buffered Saline without calcium and magnesium Sigma D8537
RPMI1640 no indicator dye Gibco/Life Technologies 11835-063
(alternatively RPMI1640 with indicator dye) (Sigma) (R8758)
Fetal Bovine Serum (FCS) Sigma F7524
4´,6-diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific 62247
Atto647N-SIP-N3-Oligo Microsynth or IDT Custom made
(Atto647N-5′-TCA GTT CAG GAC CCT CGG CT-3′-N3)
BBQ650-Quencher Microsynth or IDT Custom made
(5′-AGC CGA GGG TCC TGA ACT GA-3′-BBQ650 oligo)
Click-iT® DIBO-succinimidyl ester (DIBO-SE) Thermo Fisher Scientific C10414
Alternative: DBCO-NHS ester Kerafast (via Biozol) FCC009
ZEBA desalting columns MWCO 7 kDa Thermo Fisher Scientific 89882
ZEBA desalting columns MWCO 40 kDa Thermo Fisher Scientific 87766
Water-free DMSO Thermo Fisher Scientific D12345
(Dimethylsuloxide)
Pluronic® F-127 Thermo Fisher Scientific P3000MP
L-glutamine (200 mM) Sigma G7513-100ML
Penicillin/Streptomycin (10,000 Units/ml, 10,000 μg/ml) Gibco/Life Technologies 15140-122
1M HEPES Gibco/Life Technologies 15630-056
β-Mercaptoethanol (cell culture grade, 50 mM in PBS) PAN Biotech P07-05100
Water BioPerformance certified Sigma W3513-1L
(cell culture grade ddH2O)
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Table 2. Necessary equipment
Equipment Company Purpose
Centrifuge ‘Allegra X-15R’ Beckman-Coulter Centrifugation of 50 ml tubes, 15 ml tubes, and
V-bottom plates
Neubauer counting chamber Superior Marienfeld Cell counting
0.100 mm; 0.0025 mm2
Corning storage bottle (#430518) and Corning Sterile filtration and storage of solutions
0.22 μM sterile filter (#431118)
Sterile bench ‘Mars Safety Class 2’ Scanlaf Performance of all aseptic procedures
LSR Fortessa SORP (#647800) BD Flow cytometric analysis of single-cell
suspensions
96-well V-bottom plate (651 180) Greiner bio-one Sample preparation for flow cytometry
50 ml tubes (#352070) Falcon Centrifugation of cell suspensions
15 ml tubes (#188271) Greiner bio-one Centrifugation of cell suspensions
Serological pipettes (#606180) Greiner bio-one Pipetting
FACS tube (#352008) Corning Regular FACS tubes for the acquisition of
single-cell suspensions
100 μm filter for 50ml tubes (#542000) Greiner bio-one Isolation of single cells from tissues
40 μm filters for 50ml tubes (#542040) Greiner bio-one Generation of single-cell suspensions from
lymphoid tissues by passive filtration
Pestles (#309658) BD Passage of organ material via 100 μm filters
6-well plates (#140675) Thermo Scientific Storage of organs & mechanical tissue disruption
Milli-Q Advantage A10 Millipore On-demand provision of Millipore water (ddH2O)
Waterbath GFL 1086
1.5 ml reaction tubes Greiner bio-one 616261
2.0 ml reaction tubes Greiner bio-one 623201
1.3 Step-by-step sample preparation glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml strep-
tomycin, and 50 μM β-mercaptoethanol. Dependent of your cells
1.3.1 Preparation of stocks and solutions of interest and the protein or antibody to study, this media formu-
lation can be adapted to your needs.
DAPI
Dissolve DAPI 4´,6-diamidino-2´-phenylindole dihydrochloride DIBO-SE solution
(DAPI) in cell culture grade water to create a 1 mg/ml stock solu- Solubilize 1 mg of DIBO-SE (MW(DIBO-SE) = 488.5 g/mol) in
tion. Store the solution protected from light at 4°C. Dilute the 1023 μl water-free DMSO (final concentration 2 nmol/μl) and
stock solution 1:10,000 in FACS buffer (PBS+2% FCS) to create a store small aliquots (e.g., 2.5 μl) at –20°C. Avoid freeze-thaw-
working solution for DAPI staining of cells directly before acquisi- cycles.
tion at the flow cytometer.
Atto647N-SIP-N3-Oligo
Solubilize this custom-made Oligo in a concentration of 100 μM
FCS in cell culture grade H2O and store small aliquots (e.g., 10 μl) at
Quickly thaw FCS at 37°C in a water bath. Once completely –20°C. Avoid freeze-thaw-cycles.
thawed, incubate for 15 min at 42°C in the water bath to destroy
complement activity. Directly filter the warm FCS through a sterile BBQ650-Quencher
0.22 μmmembrane (Corning #431118) into a sterile storage bot- Solubilize this custom-made Oligo in a concentration of 100 μM
tle (Corning #430518) and aliquot into 50 ml portions. Use asep- in cell culture grade H2O and store small aliquots (e.g., 10 μl) at
tic techniques during the whole procedure. Aliquoted FCS must –20°C. Avoid freeze-thaw-cycles.
be stored at –20°C. Avoid freeze-thaw cycles.
FACS buffer 1.3.2 Preparation of protein or antibody solution
Add 2% FCS (v/v) to Phosphate buffered saline solution (PBS).
For the labeling of your protein or antibody of interest, it is
RPMI+5% FCS (culture medium) important to remove all potentially interfering substances. These
Add 5% FCS (v/v) to RPMI1640 medium without indicator dye. If include glycine, glycerin, and azide ions as these substances can
DC are in the focus of analysis, further supplement with 2 mM L- directly react with succimidyl ester group of the DIBO-SE linker.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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Eur. J. Immunol. 2023;53:2249925 Direct analysis of internalization of proteins (including antibodies) into mouse DC 9 of 96
The optimal concentration for labeling of antibodies is 1 mg/ml; 21. Add 2.5 μl DIBO-SE solution (2 nmol/μl in water-free
other proteins should have the same molarity. In case your pro- DMSO).
tein or antibody concentration is too low, it can be increased 22. Mix immediately and very well.
by an appropriate volume reduction device. For antibodies, Mil- 23. Incubate the reaction mixture for 2 hours at 4°C.
lipore/Amicron MWCO 50 kDa columns (Amicron® Ultra 4 cen-
trifugal filters, Millipore, UFC805096) might be used. However, Now, the excess of DIBO-SE and the unwanted small reac-
this might lead to a loss of antibody or protein of up to 50% due to tion products need to be removed using a ZEBA desalting column
membrane adsorption. Furthermore, these devices are not sterile MWCO 7kDa.
and may additionally contain glycerin. The removal of unwanted
24. Break the cap at the bottom of the desalting column and
salts, buffer components and organic compounds can be achieved
insert the column in an appropriate collection tube.
by buffer exchange columns (here for antibodies or bigger pro-
25. Open the cap on top of the column slightly.
teins use ZEBA desalting columns MWCO 40 kDa to exchange best
26. Centrifuge it for 1 min at 1500 × g and 4°C.
for plain PBS). The maximum volume for this buffer exchange
27. Discard the flow through.
column is 100 μl.
28. Pipet 300 μl of PBS onto the column and allow it to sink in.
1. Break the cap at the bottom of the desalting column and 29. Centrifuge it for 1 min at 1500 × g and 4°C.
insert the column in an appropriate collection tube. 30. Discard the flow through.
2. Open the cap on top of the column slightly. 31. Pipet 300 μl of PBS onto the column and allow it to sink in.
3. Centrifuge it for 1 min at 1500 × g and 4°C. 32. Centrifuge it for 1 min at 1500 × g and 4°C.
4. Discard the flow through. 33. Discard the flow through.
5. Pipet 300 μl of PBS onto the column and allow it to sink in. 34. Pipet 300 μl of PBS onto the column and allow it to sink in.
6. Centrifuge it for 1 min at 1500 × g and 4°C. 35. Centrifuge it for 2 min at 1500 × g and 4°C.
7. Discard the flow through. 36. Discard the flow through and the collection tube.
8. Pipet 300 μl of PBS onto the column and allow it to sink in. 37. Remove any liquid on the outside of the column using a tis-
9. Centrifuge it for 1 min at 1500 × g and 4°C. sue.
10. Discard the flow through. 38. Place the column in a fresh collection tube.
11. Pipet 300 μl of PBS onto the column and allow it to sink in. 39. Directly pipet the sample (in not more than 100 μl) onto the
12. Centrifuge it for 2 min at 1500 × g and 4°C. column and allow it to sink in.
13. Discard the flow through and the collection tube. 40. Pipet another 15 μl of PBS as stacker volume onto the col-
14. Remove any liquid on the outside of the column using a tis- umn.
sue. 41. Centrifuge it for 2 min at 1500 × g and 4°C.
15. Place the column in a fresh collection tube. 42. Flow through of about 75 μl contains your antibody or
16. Directly pipet the sample (in not more than 100 μl) onto the protein in PBS in approximately the same concentration as
column and allow it to sink in. before.
17. Pipet another 15 μl of PBS as stacker volume onto the col-
umn.
18. Centrifuge it for 2 min at 1500 × g and 4°C. 1.3.4 Click chemistry reaction of DIBO-SE coupled antibody
19. Flow through of about 100 μl contains your antibody or or protein with the Atto647N-SIP-N3 oligo
protein in PBS in approximately the same concentration as
before buffer exchange. Next, the alkyne of the attached DIBO-SE linker will be coupled
with the azide group of the Atto647N-SIP-N3 oligo. This attaches
the fluorophore for detection to your protein or antibody of inter-
1.3.3 Coupling of the DIBO-SE linker to the protein or
est. Therefore, the reaction will be performed with a twofold
antibody of interest
molecular excess of the oligo (stock: 100 μM equals 0.1 nmol/μl).
If you start with 75 μg of antibody (0.5 nmol), 10 μl Atto647N-
For the coupling process, a tenfold molecular excess of DIBO-
SIP-N3 oligo (1 nmol of the 100 μM stock).
SE is used. With its succimidyl ester group, it can react with
primary amines found in the lysine residues of the protein. An 43. If you started with 75 μg of antibody (equals 0.5 nmol), add
IgG molecule displays a molecular weight 150 kg/mol or 150 10 μl of Atto647N-SIP-N3 oligo (1 nmol of the 100 μM stock
μg/nmol. Therefore, to label 75 μg antibody (75 μl with 1 solution).
mg/ml), which equals 0.5 nmol, 5 nmol DIBO-SE are needed. The 44. Mix immediately and very well.
stock solution is 2 nmol/μl, so 2.5 μl DIBO-SE solution is needed. 45. Incubate the reaction mixture for 16 hours at 4°C.
For other proteins, the volumes have to be adjusted. 46. Top-up the volume to 85 μl with PBS.
20. Transfer 75 μl of antibody solution (1 mg/ml, 4°C) into a 1.5 To remove any unbound oligo, a separation using a ZEBA
ml reaction tube. buffer exchange column MWCO 40 kDa is performed.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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10 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
47. Break the cap at the bottom of the desalting column and 71. Wash the cells three times with FACS buffer as for a usual
insert the column in an appropriate collection tube. staining.
48. Open the cap on top of the column slightly. 72. Optional: Stain the single-cell suspension with additional
49. Centrifuge it for 1 min at 1500 × g and 4°C. antibodies to identify your populations of interest (e.g.,
50. Discard the flow through. DC subsets, see “Guidelines for DC preparation and flow
51. Pipet 300 μl of PBS onto the column and allow it to sink in. cytometry analysis of mouse lymphohematopoietic tis-
52. Centrifuge it for 1 min at 1500 × g and 4°C. sues” [9].
53. Discard the flow through. 73. Analyze the cells at your flow cytometer of choice (For the
54. Pipet 300 μl of PBS onto the column and allow it to sink in. Atto647N dye, a classical APC/Alexa647 detection is recom-
55. Centrifuge it for 1 min at 1500 × g and 4°C. mended, e.g., 640 nm laser line and a 670/14 nm band pass
56. Discard the flow through. filter).
57. Pipet 300 μl of PBS onto the column and allow it to sink in.
58. Centrifuge it for 2 min at 1500 × g and 4°C.
59. Discard the flow through and the collection tube.
60. Remove any liquid on the outside of the column using a tis- 1.3.6 Titration of the labeled BBQ650-quencher
sue.
61. Place the column in a fresh collection tube. After identification of the maximum staining for the outside of
62. Directly pipet the sample (in not more than 100 l) onto the your cells with the Atto647N-SIP-labeled protein or antibody, aμ
column and allow it to sink in. titration of the BBQ650-quencher is needed to ensure complete
63. Pipet another 15 l of PBS as stacker volume onto the col- quenching of the signal caused by labeled protein or antibodyμ
umn. outside the cells and to get an idea about the minimum amount
64. Centrifuge it for 2 min at 1500 × g and 4°C. of BBQ650-quencher needed. Therefore, the cells are labeled with
65. Flow through of about 85 μl contains your antibody or pro- the amount of labeled antibody, which gives the maximum signal
tein in PBS. We recommend to fill-up the volume to 100 l and different amounts of the BBQ650-quencher oligo are testedμ
to reach a concentration of about 0.5 mg/ml. to ensure complete quenching. As the BBQ650-quencher can only
access the cell surface, it is of utmost importance to keep the cells
and all solutions at 4°C.
1.3.5 Titration of the labeled antibody
74. Prepare splenic single-cell suspensions (or single-cell suspen-
Before starting with the internalization assay, we recommend to sion from your organ of interest) by digestion with Colla-
perform a titration of your antibody or protein. Thereby you can genase D and DNAse I as described in “Guidelines for DC
maximize your signal and ensure specificity. The titrations are per- preparation and flow cytometry analysis of mouse lym-
formed independently starting with the fluorescently labeled pro- phohematopoietic tissues” [9].
tein or antibody. Therefore, we recommend to use the volumes 75. Re-suspend the cells in the appropriate volume of RPMI+5%
and cell numbers that you plan to use in the assays (e.g., 50 μl of FCS medium (e.g., if the internalization into 5×106 cells in
cells in a 96-well plate). As we do not measure internalization yet, 50 μl is planned, re-suspend the cells in a concentration of
it is important to incubate the cells at 4°C, so all media and buffers 200×106 cells/ml to seed 25 μl).
should be already at 4°C and centrifuges should be pre-cooled. 76. Seed 25 μl of the single-cell suspensions in each well (5×106
cells/well).
66. Prepare splenic single-cell suspensions (or single-cell suspen- 77. Add 25 μl of your Atto647N-SIP-labeled antibody or protein
sion from your organ of interest) by digestion with Col- with the previously determined antibody concentration
lagenase D and DNAse I as described in “Guidelines for (from 1.3.5) diluted in RPMI+5% FCS.
DC preparation and flow cytometry analysis of mouse 78. Incubate for 30 min at 4°C.
lymphohematopoietic tissues” [9] containing protocols for 79. Wash the cells three times with FACS buffer (4°C) as for a
the generation of DC containing single-cell suspensions from usual staining.
spleen, lymph nodes, and thymus. 80. Add different amounts of the BBQ650-quencher (diluted in
67. Re-suspend the cells in the appropriate volume of cold FACS buffer). The usual needed range is around 1 μM
RPMI+5%FCS medium (e.g., if the internalization into (stock 100 μM).
5×106 cells in 50 μl is planned, re-suspend the cells in a 81. Optional: Stain the single-cell suspension with additional
concentration of 200×106 cells/ml to seed 25 μl). antibodies to identify your populations of interest (e.g., DC
68. Seed 25 μl of the single-cell suspensions in each well (5×106 subsets, see “Guidelines for DC preparation and flow
cells/well). cytometry analysis of mouse lymphohematopoietic tis-
69. Add 25 μl of your Atto647N-SIP-labeled antibody or protein sues” [9]).
in different concentrations diluted in RPMI+5% FCS. 82. Analyze the cells at your flow cytometer of choice (For
70. Incubate for 30 min at 4°C. the Atto647N dye, a classical APC/Alexa647 detection is
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
 15214141, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/eji.202249925 by Universitätsbibliothek Mainz, Wiley Online Library on [07/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Eur. J. Immunol. 2023;53:2249925 Direct analysis of internalization of proteins (including antibodies) into mouse DC 11 of 96
recommended, e.g., 640 nm laser line and a 670/14 nm band 91. Optional: Stain the single-cell suspensions with additional
pass filter). antibodies to identify your populations of interest (e.g., DC
subsets, see “Guidelines for DC preparation and flow
Here, the minimum amount of BBQ650-quencher needed to cytometry analysis of mouse lymphohematopoietic tis-
quench the maximum fluorescence of the Atto647N-SIP-labeled sues” [9]).
protein or antibody is determined. We recommend for the inter- 92. Analyze the cells at your flow cytometer of choice (For the
nalization to use two- to fivefold more of the BBQ650-quencher Atto647N dye, a classical APC/Alexa647 detection is recom-
as determined in this titration (incubation during the internaliza- mended, e.g., 640 nm laser line and a 670/14 nm band pass
tion later might lead to upregulation or transport of the target of filter).
your Atto647N-SIP-labeled protein or antibody to the cell surface.
By applying an excess of the BBQ650-quencher, you ensure the 1.4 Data analysis
highest specificity of the internalization assay.
To demonstrate the possibilities of this assay, we performed an
exemplary internalization assay with three different antibodies
1.3.7 Internalization assay (namely DEC205, only found on cDC1, DCIR2, only found on
cDC2, and FcγRIV, found on both cDC subsets). Therefore, we
For this assay, some controls are needed. We at least recommend investigated the internalization into cDC1 and cDC2 (Fig. 2). The
using a fluorescence-minus-one (FMO) control, a similarly labeled identification of the DC subsets was performed as described in
isotype control as well as samples stored at 4°C (without any “Guidelines for DC preparation and flow cytometry analysis of
incubation at 37°C). Additionally, we also recommend to block mouse lymphohematopoietic tissues” [9]. FlowJo v10.8 (BD)
unwanted Fc/Fc receptor interactions by applying blocking anti- was used to extract the mean fluorescence intensity values for
bodies for Fc RIIB/III (common Fc block, clone 2.4G2) as well as cDC1 and cDC2 DC. Next, the mean fluorescence intensity of theγ
FcγRIV (also known as CD16.2, clone 9E9). As the samples will sample without Atto647N-SIP-labeled antibody was subtracted
be incubated for different timespans, we recommend to perform (giving the MFI values). In Figure 2, we compare the change of
this assay in 1.5 or 2 ml reaction tubes. these MFI values over the different incubation periods and also
marked the maximum fluorescence intensity of the extracellular
staining with the Atto647N-SIP-labeled antibodies (strictly kept
83. Prepare splenic single-cell suspensions (or single-cell suspen- on ice and not quenched). Thereby, two different classes of recep-
sion from your organ of interest) by digestion with Colla- tors can be determined: 1) DEC205 as well as FcγRIV is internaliz-
genase D and DNAse I as described in “Guidelines for DC ing more antibody over time as could be stained extracellularly, if
preparation and flow cytometry analysis of mouse lym- samples were kept on ice only) providing evidence for a replenish-
phohematopoietic tissues” [9]. ment of internalizing receptors by recycling receptors or receptors
84. Re-suspend the cells in the appropriate volume of
+ stored intracellularly and 2) DCIR2 is internalizing less antibodyRPMI 5%FCS medium (e.g., if the internalization into
× than bound on the cell surface (at least over the 2 hours incuba-5 106 cells in 50 μl is planned, re-suspend the cells in a
× tion period).concentration of 200 106 cells/ml to seed 25 μl).
85. Seed 25 μl of the single-cell suspensions in the 1.5 or 2 ml
reaction tubes (5×106 cells/well). 1.5 Pitfalls
86. Add 25 μl of your Atto647N-SIP-labeled antibody or pro-
tein with the two- to tenfold amount of the previously Problem: No signal from the labeled protein or antibody
determined antibody concentration (from 1.3.5) diluted in within the titration.
RPMI+5% FCS to also be able to observe a “top-up”-effect Potential solution:
over time. Ensure the expression of the antibody target or protein binding
87. Incubate the samples for the wanted timespans (e.g., 0, 10, partner (e.g., by staining with a classical antibody).
30, 60, or 120 min; dependent on your receptor) at 37°C Ensure the correct labeling of your antibody with the DIBO-
and 5% CO2 (or your preferred culture conditions); non- SE and the subsequent Atto647N-SIP-N3 oligo. For this, there are
internalized controls are strictly stored at 4°C. different possibilities. First, for classical antibodies it is possible to
88. After the end of the incubation, immediately cool down the use compensation beads, such as UltraComp eBeadsTM Plus Com-
sample (best by transferring to wet ice and filling up with pensation Beads (Thermo Fisher Scientific), and ensure, if you can
10 to 20 times of the starting volume with cold (4°C) FACS label these beads using the Atto647N-SIP-N3-labeled antibodies.
buffer. Second, which is also possible for Atto647N-SIP-N3-labeled pro-
89. Wash the cells three times with FACS buffer (4°C) as for a teins, the degree of functionalization can be measured on a Nan-
usual staining. oDrop 1000 UV-VIS spectrophotometer.
90. Add two- to fivefold amount of the BBQ650-quencher as Alternatively, also other reactive groups or linkers may
determined in 1.3.5 (diluted in cold FACS buffer). be used. These include Click-iT® DIBO-amine (reacting with
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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12 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Figure 2. Exemplary data for the internalization of Atto647N-SIP-labeled antibodies including quenching of extracellular fluorescence. Splenic
single-cell suspension of female, ten weeks old C57Bl/6J mice were generated including Collagenase IV/DNAse I digestion and erythrocyte lyses.
Each 5×106 cells were incubated with 10 μg/ml Atto647N-SIP-αDEC205, Atto647N-SIP-αDCIR2, or Atto647N-SIP-αFcγRIV antibodies in RPMI+5 %FCS
for 0, 5, 10, 30, 60, or 120 min at 37°C and 5% CO2 and quenched with the BBQ650-quencher oligo. Additional controls included samples without
antibody staining as well as an additional sample, which was individually incubated with the same antibody amounts and strictly kept on ice (for
the maximum extracellular staining). After staining with the cell identification antibodies, the cells were acquired utilizing a BD LSR Fortessa SORP
flow cytometer. Data were analyzed by FlowJo v10.8 (BD) to evaluate the Atto647N fluorescence for cDC1 and cDC2 DC. The panels depict the mean
fluorescence intensity for 1) cDC1 (left panel) and 2) cDC2 DC (right panel) in dependency of the internalization period. The dotted lines mark the
mean fluorescence intensity of the control sample without internalization and quenching.
carboxylic acids) and Click-iT® DIBO-maleimide (reacting with The labeling technique can be also utilized to label bigger
thiols). structures, such as nanoparticles or exosomes to analyze their
uptake. Furthermore, the Atto647N dye is also compatible to
Problem: Antibody or protein of interest is not labeled. microscopy to visualize the endocytosed antibodies or proteins.
Potential solution: Additonally, the method is in principle also suited to study
Ensure that no other substances including primary amines and endocytosis in vivo, but high amounts of the conjugate will be
no azide groups or ions are present during the reaction with the needed. We would suspect the need for the injection of 10 to
DIBO-SE linker. Furthermore, ensure that there are enough lysine 100 μg per mouse (will strongly depend on the receptor of
residues free for the coupling to the DIBO linker in your anti- interest and the antibody or protein used). Shortly after sacrifice,
body (normally the case) or your protein. Furthermore, ensure the samples need to be cooled to 4°C to avoid the continuation
the absence of substances inhibiting the copper-free click chem- of endocytosis before quenching ex vivo with the respective
istry within the reaction with the Atto647N-SIP-N3 oligo. quencher.
Problem: During the titration of the BBQ650-quencher, only a
partial quenching is possible. 2 Mitochondrial function assessment of
Potential solution: mouse Fms-like tyrosine kinase 3 ligand
Ensure that the sequence of the Atto647N-SIP-N3 oligo is comple- derived-DC using an extracellular flux
mentary invers to the used BBQ650-quencher. Furthermore, it is analyzer
important to ensure the right orientation of the BBQ650-quencher
to the Atto647N-dye (ensured with the given sequences here), so 2.1 Introduction
if the dye is 5’, the BB650 BHQ needs to be 3’.
The labeling and the subsequent washing steps were not In the last years, most research articles interrogating metabolic
performed at 4°C and a partial internalization was taking place. adaptation of dendritic cells (DC) to microbial stimuli or envi-
In this case, ensure to keep all buffers, reagents, and centrifuges ronmental cues, made use of in vitro DC differentiated with
cold. granulocyte-monocyte colony-stimulating factor (GM-CSF) [10].
The amount of labeled antibody is too high or the amount used However, the predominant presence of macrophage-like cells and
quencher is too low. Use less labeled antibody or more quencher. inflammatory DC in this culture setting prevents the extrapolation
of results to bona fide DC found in physiological conditions [11].
For instance, the presence of other cell types in GM-CSF DC cul-
1.6 Top tricks tures have led to confounding results implying that the sustained
upregulation of glycolysis upon Toll-like receptor stimulation, is
Here, we describe the analysis of the uptake of one antibody or coupled to DC full activation [12]. However, this could not be
protein. It can be extended to two or more proteins at the same demonstrated in other DC cultures lacking inducible nitric oxide
time. Therefore, you just need to choose a second combination of synthetase (iNOS) expression or in GM-CSF DC prepared from
a DNA sequence, fluorophore, and a compatible quencher. iNOS KO bone marrows (non-published own data), interrogating
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 Mitochondrial function assessment of mouse Fms-like tyrosine kinase 3 ligand derived-DC 13 of 96
the real contribution of late glycolytic reprogramming to immuno- Table 3. List of reagents used for Cell Mito Stress assay
genic DC activation.
Reagent or resource Company Catalog
To date, the low number of ex vivo DC that can be isolated from
lymphoid organs does not fulfill the minimum input required for Bone marrow isolation
evaluating metabolic processes by conventional methods such Bone marrow cells - -
as mass spectrometry. Besides, classical biochemical assays that RPMI 1640 Thermo Fisher 61870
directly measure the consumption of substrates, have become Scientific
inconvenient and obsolete due mainly to the use of radioactive Fetal calf serum (FCS) Biochrom S0115
isotopes. heat-inactivated
2-Mercaptoethanol Gibco 31350
Therefore, to overcome these limitations, we provide here
Mouse FLT3L-flag culture Made in-house N/A
a protocol for assessing the mitochondrial function of mouse supernatant
bone marrow-derived DC (BMDC) with an extracellular flux Petri dishes 10 cm, sterile Sarstedt 821473001
analyzer. 50 ml tubes Sarstedt 62547254
As a model for studying in vitro DC metabolism, we propose Lipopolysaccharid Sigma Aldrich L3024
using the Fms-like tyrosine kinase 3 ligand (FLT3L)-DC culture, aus Escherichia
which yields a mix of various DC sub-populations (i.e., classi- coli O111:B4
cal DC1, classical DC2, and plasmacytoid DC) resembling in vivo Cell Mito stress assay
counterparts and thus outperforms the GM-CSF culture in the XFe96 FluxPak including Agilent Technologies 102416-100
evaluation of DC immunological and metabolic traits [13]. sensor cartridges, cell
In addition, the use of the Seahorse XFe96 analyzer to monitor culture microplates, and
calibrant solution
mitochondrial function surpasses the classical methods in several
XF Seahorse RPMI assay Agilent Technologies 103576-100
aspects. First, the cell number required is lower than that of other medium, pH 7.4
techniques, and multiple conditions can be tested in parallel due Poly-L-lysine 0.01% Sigma Aldrich P4707
to the use of a 96-well microplate. Second and most importantly, solution
it is possible to do real-time measurements of the oxygen con- Sodium Pyruvate 100 mM Sigma Aldrich S8636
sumption rate (OCR), an indicator of oxidative phosphorylation Glucose Carl Roth HN06.2
(OXPHOS), in live cells. Third, the ease of Seahorse’s operation L-Glutamine Sigma Aldrich G3126
and the simplicity and quickness of data analysis turn the Sea- Oligomycin Sigma Aldrich 75351
horse XFe96 analyzer into a more user-friendly approach than, Antimycin A Sigma Aldrich A8674
for instance, mass spectrometry. Carbonyl cyanide-p- Cayman chemicals 15218
Noteworthy, we incorporated a normalization step within the trifluoromethoxyphenyl
hydrazone
assay workflow based on in-situ cell counting with the integrated
(FCCP)
Cytation 5 multi-mode reader. Data normalization is fundamen- Rotenone Cayman chemicals 13995
tal during a mitochondrial stress test since OCR heavily correlates Hoechst 33342 (20 mM) Thermo Fisher 62249
to the number of cells per well, and stimulations or treatments scientific
can induce either cell proliferation and/or cell death to a vari- Ultrapure distilled water Thermo Fisher 10977-049
able extent. Albeit protein amount determination upon Seahorse scientific
XF analysis has been widely used for the sake of inter-well nor-
malization; it is inaccurate, time-consuming, and does not allow
for downstream applications. Cytation 5 fixes these issues by per- macological manipulation of DC metabolism as new therapeutic
forming rapid automated cell counting of fluorescently-labeled strategy.
nuclei followed by straight-forward normalization with the Sea-
horse Wave software.
Our main focus lies on mitochondrial metabolism due to the 2.2 Materials
proven relevance of this organelle for DC differentiation and
function [10]. In brief, during the mitochondrial stress test, cells
are sequentially treated with compounds that target the electron 2.2.1 Reagents
transport chain (ETC) or disrupt the mitochondrial membrane
potential. As a result, various parameters such as the basal A detailed list of necessary reagents for FL-DC generation and Cell
respiration and maximal respiration rates can be estimated, and Mito Stress assay is provided in Table 3.
therefore the overall mitochondrial metabolism can be inferred
[14].
Finally, gaining accurate knowledge of DC metabolism would 2.2.2 Equipment
contribute to clarifying the role of specific metabolic pathways for
DC activation and function, and thereby would enable the phar- Necessary equipment is listed in Table 4.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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14 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 4. List of necessary equipment * Note: The coated cell culture microplate can be stored in the
fridge overnight, but make sure to warm it up to RT before
Equipment Manufacturer Model
using it.
Laminar flow hood Thermo Fisher Scientific Herasafe 2030i
Centrifuge Thermo Fisher Scientific Megafuge ST Seeding the cells:
Plus Series
5
Inverted Zeiss Primovert 1. Add 40 μl/well (1×10 FL-DC/well) of the FL-DC cell suspen-
microscope sion into a PLL-coated cell culture microplate. Complete by
Agilent Seahorse Agilent technologies adding 40 μl of growth medium (made by complete medium
XFe96 analyzer supplemented with 15% FLT3L supernatant (176 ng/ml)), LPS
BioTek Agilent technologies (100 ng/ml final concentration in growth medium), or the
Instrument’s nitric oxide (NO) donor DETA-NONOate (DETA, 200 μg/mL
Cytation 5 in growth medium). Hence, the final volume per well will be
Incubation shaker Infors HT Minitron 80 μl. An example of a cell plate layout for each condition is
CO2 incubator Thermo Fisher Scientific Heracell 240i shown in Figure 3.
* Note: The addition of FLT3L at the same concentration
2.3 Step-by-step sample preparation used for bone marrow differentiation is recommended when
overnight stimulation is required (e.g. LPS), as it was shown
to increase DC survival.
2.3.1 Generation of FLT3L-DC (FL-DC) cultures from mouse
2. Add 80 μl/well of only complete medium supplemented with
bone marrow
15% FLT3L supernatant (176 ng/ml; no cells) into the back-
ground wells. In general, at least 4 wells (i.e. 1A,1H,12A, 12H)
1. Isolate BM cells from mouse femurs and tibias.
× are used as a background/correction well (Fig. 3).2. Seed 15 106 BM cells into a 10 cm-dish in a total volume
3. Examine the plate under an inverted microscope to ensure the
of 10 ml of complete medium supplemented with 15% super-
cells are plenty settled at the bottom of the plate.
natant of an FLT3L-producing cell line (176 ng/ml)[13].
4. Incubate the cell culture microplate in a 37°C humified 5%
* Note: Henceforth, complete medium consists of RPMI 1640
CO2 incubator for 24 h.
supplemented with 2-mercaptoethanol (50 μM), penicillin
(100 u/ml)/streptomycin (0.1 mg/ml), and FCS (10% v/v). Hydrate cartridge:
The optimal working concentration of FLT3L needs to be
determined empirically upon FLT3L new batch production. 1. Open an XFe96 sensor cartridge box and separate the utility
3. Place the dish in a humified cell incubator (37°C, 5% CO2) and plate and sensor cartridge (Fig. 4A).
culture the FL-DC for 9 days. 2. Place the sensor cartridge upside down next to the utility
4. Harvest the FL-DC on day 9 into a 50 mL tube and centrifuge plate and add 200 μl/well of sterile XF Calibrant to the utility
the cell suspension at 400g at 4°C for 7 min. plate.
* Note: 8–12 × 106 cells/dish are expected after 7–9 days of * Note: Keep the XF Calibrant solution sterile
culture 3. Put the sensor cartridge back onto the utility plate, submerging
5. Count the cells and resuspend the cell pellet in an appropriate the sensors in the calibrant carefully.
volume of complete medium supplemented with 15% FLT3L 4. Place them in a 37°C non-CO2 incubator overnight. To prevent
supernatant (176 ng/ml). evaporation of the water, verify that the incubator is properly
6. Adjust the concentration to seed 40 μl of the cell suspension humidified.
at a density of 1×105 FL-DC/well into an XF96 cell culture
microplate (see section “seeding the cells”). Preparing the Seahorse extracellular analyzer:
1. Turn on the Agilent Seahorse XFe96 Analyzer, and let it warm
up overnight. The analyzer needs to warm up for at least 5 h
2.3.2 Seahorse cell mito stress assay before starting the assay.
* Note: Make sure the heater is ON and the software recog-
Day before assay nizes the instrument.
Preparing the XF96 cell culture microplate:
Day of assay
1. Coat the XF96 cell culture microplate with 20 μl of 0.01% Preparing the assay medium and washing the cells:
poly-L-lysine (PLL) and incubate in a non-CO2 incubator for at
least 1 h. 1. Prepare fresh Mito stress assay medium as shown in Table 5.
2. Carefully, rinse twice the PLL with sterile ultrapure distilled The final volume depends on how many wells are seeded. We
water and dry it under the hood for at least 2 h before seeding recommend using the Seahorse XF RPMI medium ready to use
cells. (low buffered media, pre-adjusted to pH 7.4).
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 Mitochondrial function assessment of mouse Fms-like tyrosine kinase 3 ligand derived-DC 15 of 96
Figure 3. Illustration of a cell culture microplate layout for each condition.
* Note: The concentration of glucose should also be deter- by gently adding 180 μl of warmed XF assay medium using a
mined experimentally and adjusted to the cell type of multichannel pipette. Repeat this washing step.
interest. * Note: The washing steps should be performed carefully to
2. Once prepared, warm the XF assay medium in a water bath at avoid disrupting the cell monolayer
37°C. 5. Spin down the cell culture microplate slightly in a centrifuge
3. Retrieve the cell culture microplate from the CO2 incubator at RT to facilitate the cell adherence to the bottom of the well.
and examine the cells under an inverted microscope to confirm Set the acceleration and deceleration to 5 and centrifuge at
cell health morphology, confluence (uniformity), and purity 250g for 1 min.
(no contamination). 6. Examine the cells once again under an inverted microscope to
4. Remove the XF assay medium from the water bath and wash ensure that cells adhered to the bottom of the wells and to
the cell culture growth medium in the cell culture microplate verify the presence of consistent monolayer along groups.
Figure 4. Photographs of XFe96 sensor cartridge and utility plate. A) Sensor cartridge and utility plate used for an XF Cell Mito Stress assay. B)
Representative layout illustrating the corresponding compound to be added into each injection port of the hydrated Sensor cartridge prior to its
calibration.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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16 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 5. XF assay medium
Reagent Stock Final concentration Volume
Seahorse XF RPMI assay medium, pH 7.4 (without - - 50 ml
phenol red, glucose, pyruvate, and L-glutamine)
Glucose 1 M 2.5 mM 125 μl
Pyruvate 100 mM 1 mM 500 μl
L-glutamine 200 mM 2 mM 500 μl
7. Place the cell culture microplate into a 37°C non-CO2 incuba- run the XF Cell Mito Stress Test on the Agilent Seahorse
tor for 45 min to 1 h before the assay. XFe96 Analyzer.
5. Set up the standard protocol as shown in Table 7.
Preparation of the mitochondrial modulators and protocol setup 6. After setting the conditions for the assay, press the button
“Run assay”.
1. Prepare the 10× injection solutions for each modulator of 7. Follow the instructions on the screen and place the hydrated
mitochondrial respiration using an assay medium. Stocks cartridge without the lid on the Seahorse XFe96 analyzer for
and final concentrations for each compound are detailed in calibration and equilibration. The procedure lasts approxi-
Table 6. The volume of the solution for each compound sug- mately 25 min.
gested is 3 mL for a whole cartridge. 8. Once the calibration is finished, replace the utility plate for
2. Remove the hydrated cartridge from the non-CO incuba- the cell culture microplate previously placed in the 37°C non-2
tor and add the appropriate volume of each freshly prepared CO2 incubator.
injection solution into ports A, B, and C as indicated in Figure 9. After the XF Cell Mito stress assay is completed, remove the
4B. cell culture microplate and determine the cell counts by using
* Note: A nuclear staining dye such as Hoechst 33342 is the BioTek Instrument’s Cytation 5.
added on port C in this step to normalize the raw data once 10. Keep the display open to perform normalization of the XF
the assay is completed (see section “Normalization using a raw data.
BioTek Instrument’s Cytation 5”).
3. Inspect the sensor cartridge to verify the solutions are loaded
evenly into the ports.
4. Open Wave Desktop software and create a new assay by 2.3.3 Normalization of Agilent Seahorse XF data by nuclear
selecting the template file provided by the manufacturer to cell counting using a BioTek Cytation 5
Table 6. List of compounds and the corresponding stock and working 1. Turn on the instrument while the XF Cell Mito stress assay is
concentration running. The instrument needs time to warm up.
Compound Stock Final 10× 2. Once the XF Cell Mito stress assay is completed, open “Gen5
concentration desktop software” on the Agilent Seahorse XFe96 Analyzer.
3. Transfer the cell culture microplate from the Seahorse XFe96
Oligomycin 2.5 mM in DMSO 2 μM 20 μM analyzer to the BioTek Cytation 5 instrument immediately, ver-
FCCP 2.5 mM in DMSO 1.5 μM 15 μM ifying the microplate is loaded in the correct orientation as
Rotenone 2.5 mM in DMSO 0.5 μM 5 μM indicated in the plate tray.
Antimycin A 2.5 mM in DMSO 0.5 μM 5 μM
4. Select “Fluorescent cell counting” on Gen5 Software and
Hoechst 33342 20 mM 1.7 μM 17 μM
choose the necessary wells manually.
Table 7. Setting the assay protocol
Step Compound Cycle Measurement details Duration
Calibrate – – –
Equilibrate – – –
Baseline – 3 Mix-3’; Wait-2’; Measure-3’ 24’
Inject port A Oligomycin 3 Mix-3’; Wait-2’; Measure-3’ 24’
Inject port B FCCP 3 Mix-3’; Wait-2’; Measure-3’ 24’
Inject port C Rotenone 3 Mix-3’; Wait-2’; Measure-3’ 24’
Antimycin A
Hoechst 33342
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 Mitochondrial function assessment of mouse Fms-like tyrosine kinase 3 ligand derived-DC 17 of 96
Figure 5. Metabolic profile of FL-DC upon LPS stimulation. FL-DC were harvested on day 9 and stimulated with 100 ng/ml of LPS or 200 μg/mL of
DETA in an XFe96 cell culture microplate for 24 h before XF Cell Mito stress assay. A) Schematic representation of XF Cell Mito Stress Test profile
illustrating the sequential injection of different mitochondrial modulators and the key parameters obtained with this assay. B) Oxygen consump-
tion rates (OCR) normalized against cell numbers from FL-DC cultured in the presence or absence of LPS and DETA, measured by extracellular
flux analysis. OCR was determined at baseline and after sequential treatment with the mitochondrial complex IV inhibitor oligomycin (Oligo),
mitochondrial uncoupler FCCP, and rotenone plus antimycin A (Rot/AA) to inhibit mitochondrial complexes I and III. C) Normalized basal OCR
(left) and quantification of maximal OCR (Max OCR, right) from FL-DC in the presence or absence of LPS or DETA. Representative results from 2
independent experiments are shown. Data shown are mean ± SD, n = 5 technical replicates. **P<0.01 was determined by one-way ANOVA. D)
Representative images for each condition obtained by BioTek Cytation5 after cell counting using Hoechst 33342. The nuclear dye was included in
the Rotenone/Antimycin Amixture on port C. Nuclear segmentation is represented by yellow contour and was obtained using Gen5 software. Scale
bars represent 1000 μm.
5. Start the cell counting. The duration of this step depends on mitochondrial respiration [15]. Oligomycin, injected first after
the number of wells previously selected. The estimated count- basal OCR measurements, inhibits the activity of the ATPase (com-
ing time is approximately 20 min. plex V) therefore decreasing OXPHOS (and OCR). FCCP, the sec-
6. Once the cell images are captured, remove the cell culture ond injection, uncouples oxygen consumption from ATP produc-
microplate. tion increasing OCR to the maximum. Therefore, this uncoupler
7. Inspect the captured images in each well and record them if agent estimates the maximum respiration rate that a cell can
necessary. achieve, afterward used to calculate spare respiratory capacity,
8. Normalize the XF raw data on the Agilent Seahorse XFe96 Ana- defined as the difference between maximal respiration and basal
lyzer using Wave software. respiration. Spare respiratory capacity is a measure of the capa-
* Note: As the BioTek Cytation 5 can read the bar code of a bility of the cell to respond to increased energy demand or cel-
specific XF cell culture microplate and both instruments are lular stress. The third injection consists of a mixture of rotenone
connected, the cell counting provided will be immediately and antimycin A, which inhibits complex I and III of the ETC,
available on the Seahorse XFe96 analyzer. respectively, and thereby results in a complete OXPHOS blockade
9. Proceed to export the Excel or Prism files generated by Wave enabling the calculation of non-mitochondrial respiration (Fig.
Software. 5A).
As previously mentioned, the Seahorse XFe96 analyzer has the
advantage of automatically calculating several parameters asso-
2.4 Data analysis ciated with mitochondrial function from Wave data by using the
Cell Mito Stress Test Report Generator.
This protocol and workflow were validated for FL-DC; however, Although DC metabolism has been widely explored using GM-
they could also be used for different cell types exhibiting varied CSF cultures, few studies have addressed it on conventional DC,
morphological characteristics in vitro. either isolated ex vivo or from FLT3L-DC cultures. In our setting,
It is highly recommended to check Agilent webpage for more we assessed OCR as a measurement of mitochondrial respiration
information about the Seahorse XFe96 analyzer before proceed- in FL-DC in the presence of LPS or DETA. As shown in Fig. 5B-
ing. C, no differences in either basal respiration or maximal oxygen
This assay consists of a sequential injection of different com- consumption rate (Max OCR) could be detected upon 24h of LPS
pounds that target distinct complexes of the ETC modulating activation. However, a blockade of the mitochondrial respiration
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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18 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
was observed when FL-DC were stimulated with DETA (Fig. 5C), Here, we adjusted the optimal cell density at 1×105 FL-
as previously reported in GM-CSF cultures [16]. In our experi- DC/well, however, it varies with different cell types depending
mental setting, we utilized bulk FLT3L-DC cultures which consist mainly on the cell size. In our experience, the optimal cell seed-
of a mixture of conventional and plasmacytoid DC. Nevertheless, ing density usually is between 1–2×105 cells/well and should
isolation of individual subpopulations from this DC culture can be result in cells distributed in the well as a monolayer at 70–90%
performed by magnetic-assisted or fluorescence-assisted cell sort- confluency.
ing, resulting then in distinct and cell type-specific mitochondrial Although we used a PLL-coated cell culture microplate to
respiration profiles. ensure cell adherence, it is possible to use different plate coat-
As we highlighted, the normalization of raw data is an impor- ing reagents such as Cell Tak, or even ready-to-use poly D-lysine
tant step to ensure accuracy and consistency in the interpreta- coated XFp Cell Culture microplates provided by Agilent.
tion of results when performing an extracellular flux analysis. When performing the washing steps on the day of assay, it
Therefore, we included in our protocol an efficient and sim- must be done carefully without touching the bottom of the well
ple way to normalize our XF raw data using Hoechst 33342, a to prevent loss of cells that might lead to significant dispersion
membrane-permeant blue fluorescent DNA stain. Combining the between replicates. Furthermore, we suggest not to remove the
in situ nuclear staining capability of Seahorse XFe96 analyzer with total volume existing in the well during this step, especially when
BioTek Instrument’s Cytation 5 system allowed us to normalize using semi-adherent cells. At the end of the washing step, cells
our results against cell numbers for all the conditions evaluated. must be in a final volume of 180 μL, critical to keep the correct
As shown in Fig. 5D, FL-DC were distributed uniformly through- concentration of each mitochondrial modulator.
out the wells for all the conditions, however upon LPS activation After washing the plate, cells need to be incubated without
they appeared disposed in typical aggregates at the bottom of the CO2 for up to 1 h to ensure a complete attachment to the bottom.
well. However, longer incubation times could substantially affect their
viability since XF assay medium has low nutrient concentration.
All ports in the hydrated cartridge need to be filled with the
appropriate volume (20, 22, and 25 μL for ports A, B and C,
2.5 Pitfalls
respectively), even for those unused. Otherwise, the injection of
the wells might be uneven, finally affecting the experimental out-
The cell seeding density represents a critical step in this assay
come. In addition, special care must be taken when loading the
and must be established beforehand. Excessive number of cells
ports. Owing to the very low volumes pipetted and the small port
can cause poor cell adhesion and inaccurate OCR measurements.
size, we advise to insert the pipette tip into the port and then dis-
In order to avoid it, it is strongly recommended to examine cell
pense the liquid avoiding to create air bubbles. At the end of the
distribution under an inverted microscope to ensure cells are
port loading process, only when small residual drops are present
adhered to the coated surface evenly and have minimal cell clus-
on top of ports, the cartridge can be gently tapped to settle them
ters. If cell loss is detected in a particular well under the micro-
to the bottom.
scope, we suggest keeping track of it in case of inconsistency in
Although all Seahorse XF reagents must be kept sterile, this
the results.
assay does not necessarily have to be performed in sterility; how-
Negative OCR values in the calibration step are feasible when
ever, it might be critical for some cell types such as DC.
trapped bubbles are present between the cartridge and the cali-
bration solution during the hydration of the cartridge.
The appropriate concentration used for each mitochondrial 3 Single cell RNA sequencing of human
modulator is also a decisive step. In our setting, we have deter- tissue DC
mined experimentally the optimal conditions by titrating each
compound in advance (Table 6). If a different cell type will be
3.1 Introduction
screened we recommend testing it as the optimal drug concentra-
tion varies according to cell type and assay medium (Tables 5 and
Most of our knowledge about dendritic cells (DC), their vari-
6). To note, commonly oligomycin and FCCP concentrations are
ous subsets, and diverse functions in health and disease came
between 0.5-2.5 μM and 0.125-2 μM, respectively.
from studies looking at whole cell populations, often by simple
microscopy, surface marker flow cytometry analyses, and bulk
mRNA sequencing technologies. While this generated very valu-
2.6 Top tricks able results, allowing us to identify specific characteristics and
functions of certain DC subsets and identification of tissue-specific
This protocol describes a setting for performing a, XF Cell Mito DC populations, these bulk technologies left a lot of questions
Stress Test for FL-DC cultures, yet different conditions might be unanswered. Single-cell analyses and sequencing technologies
needed for other cell types. Therefore, it is important not only to have since progressed rapidly and single-cell mRNA sequencing
determine the proper cell density but also the concentration of the (scRNAseq) has become an essential technology performed pretty
mitochondrial modulators experimentally. much on a daily basis, to study cellular phenotypes, functional
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
 15214141, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/eji.202249925 by Universitätsbibliothek Mainz, Wiley Online Library on [07/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Eur. J. Immunol. 2023;53:2249925 Single cell RNA sequencing of human tissue DC 19 of 96
Table 8. Reagents, chemicals, and solutions NAseq and even single-cell epigenetic analyses (scATAC-seq), and
others. These technologies allow for unbiased and simultaneous
Reagent Manufacturer Ordering number
characterization of tens of thousands of individual cells, gener-
Chemicals & Solutions ating massive data sets. This in turn comes with analytical chal-
Triton X-100 Sigma-Aldrich T9284 lenges, how to process such large amounts of data, adjust for vari-
DNA High Sensitivity PerkinElmer CLS760672 ability between tissues and species or sparsity of low-represented
Reagent Kit cell populations, etc. [19–21]. Several computational tools have
Dulbecco´s Phosphate Sigma D8537 been developed just over the past few years, facilitating the analy-
Buffered Saline ses of scRNAseq data, and new methods are being published con-
without calcium and
stantly. These analyses methods range from data normalization,
magnesium
EDTA Promega V4231 dimensionality reduction, integration of multiple data sets into
Fetal Bovine Serum Serana 0261S-FBS-SA-015 one, clustering and visualization of cell populations, differentially
(FBS) expressed genes or differentially expressed gene regulatory net-
Illumina Nextera XT kit Illumina FC-131-1096 works (regulons), to cell-to-cell interaction analyses, and many
Nextera XT DNA Library 20027213- 20027216 more [22–25].
Prep (96 Samples) The fast and massive generation of scRNAseq data studying
IDT for Illumina Nextera DC across tissues, and species, over the past years, has already
UD Indexes Set A-D improved our understanding of the vast heterogeneity of DC, their
(96 Indexes, 96 specialized functions and roles under physiological conditions,
Samples) during inflammation, infectious diseases, cancer or autoimmunity,
LIVE/DEADTM Fixable Life Technolo- L23105
and others. Here, we provide an example for scRNA sequencing of
Blue Dead Cell Stain gies
Kit DC, by indexed-SMARTseq2 scRNAseq data of human tissue DC.
RPMI 1640 HyClone SH30255.01
Sodium Azide Sigma-Aldrich 13412
3.2 Materials
states, metabolism, and even differentiation pathways of DC in 3.2.1 Reagents
infectious diseases, cancer, autoimmunity, and many other set-
tings [17, 18]. The analyses of single cells vs bulk populations A list of chemicals and solutions for flow cytometry staining is pro-
allowed us to identify novel subsets and subset functions, that are vided in Table 8. A list of antibodies required is listed in Table 10.
easily missed when studying entire tissue populations only.
With the improvement of scRNAseq technologies, these have
not just gotten cheaper, with more providers and technologies to
choose from [18], but also more accessible to a wider range of 3.2.2 Equipment
researchers. This further led to advances in technologies, such as
combining marker-informed sorting of DC populations with scR- Necessary equipment is listed in Table 9.
Table 9. Necessary equipment
Equipment Company Purpose
Centrifuge “Allegra X-15R” Beckman-Coulter Centrifugation of 50 ml tubes, 15 ml
tubes, and V-bottom plates
Illumina HiSeq 4000 Illumina Paired-end sequencing
LabChip Perkin Elmer Monitoring of length distribution of the
cDNA libraries
Sterile bench - Performance of all aseptic procedures
96-well V-bottom plate (#GN651180) Greiner Sample preparation for flow cytometry
2 ml microcentrifuge tubes Eppendorf Storage and mincing of lung tissue for
(#0030120094) digest
5 ml polystyrene flow cytometry tubes FalconTM Staining of samples for flow cytometry
(#352008)
50 ml conical tubes (#352070) FalconTM Centrifugation of cell suspensions
Serological pipettes (e.g., #GN606180) Greiner Pipetting
FalconTM 70 μm Cell Strainer, for 50 ml Corning Filtration of lung tissue, generation of
tubes (#352350) single-cell suspensions
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20 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 10. Antibodies used for Index-sorting of DC populations
Antibodies Fluorochrome Clone Manufacturer Ordering number
CADM1 Purified 3E1 MBL CM0004-3
CD1a AF700 HI149 Biolegend 300120
CD1c BV421 L161 Biolegend 331526
CD3ε BV650 SP34-2 BD Biosciences 563916
CD5 BV711 UCHT2 BD Biosciences 563170
CD11b Biotin M1/70 BD Biosciences 5533069
CD14 BV650 M5E2 BD Biosciences 563419
CD16 APC/Cy7 3G8 BD Biosciences 302018
CD19 BV650 SJ25C1 BD Biosciences 563226
CD20 BV650 2H7 BD Biosciences 563780
CD45 V500 HI30 BD Biosciences 560777
CD45RA FITC 5H9 BD Biosciences 556626
CD88 PE/Cy7 S5/1 Biolegend 344308
CD123 BUV395 7G3 BD Biosciences 564195
CD141 APC AD5-14H12 Miltenyi 130-090-907
CD163 BUV605 GHI/61 Biolegend 333616
CD169 PE 7-239 BD Biosciences 565248
CD206 PE/CF594 19.2 BD Biosciences 563780
HLA-DR BV786 L243 Biolegend 307642
FcεR1α PerCP AER-37 (CRA-1) Biolegend 334616
Anti-chicken IgY AF647 - Jackson 703-606-155
Streptavidin BUV737 - BD Biosciences 564293
3.3 Step-by-step sample preparation 1. Either:
a. Prepare single-cell suspensions from human tissue fol-
3.3.1 Preparation of stocks and solutions lowing the protocol described in “Chapter IX Mononu-
clear phagocyte phenotypes” in Cossarizza et al. (2021)
Lysis Buffer [27].
Lysis buffer is prepared as per the SMARTSeq2 protocol [26]. Or
0.2% (vol/vol) Triton X-100 + 2 U/ l RNase inhibitor. Store at b. Thaw previously prepared cells from liquid nitrogen andμ
4°C for up to 6 months. transfer into RPMI supplemented with 20% FBS.
• Incubate cells with 1 mg/ml DNase I at 37°C until all
FBS cell aggregates disappear (up to 30 min).
TM
Thaw FBS in a water bath, at 37°C. Incubate thawed FBS for 2. Incubate cells with LIVE/DEAD Fixable Blue Dead Cell
30 min at 56°C in a water bath to inactivate. Working in a sterile Stain (1:1000), in PBS, for 30 min at 4°C.
bench, filter inactivated FBS through a sterile 0.22 m mem- 3. Without washing add 5% FBS and incubate cells for 15 minμ
brane (Corning #431118) into a sterile storage bottle (Corning at 4°C, in the dark.
#430518) and aliquot into 50 ml tubes. Store aliquots at –20°C. 4. Add 2 ml of FACS buffer (if using 96-well plate add 200 μl)
Flow cytometry buffer (FACS buffer): and centrifuge at 650 G for 2 min, at 4°C.
Add 2% FBS + 2 mM EDTA (solved in PBS) + 0.05% Sodium 5. Aspirate the supernatant.
azide in 1× PBS. Store at 4°C for long-term storage or on ice for 6. Re-suspend the cell pellet in 50 μl of antibody cocktail (using
immediate use and during the experiment. the antibody panel provided in Table 10). Incubate for 30
min, at 4°C in the dark.
7. Add 2 ml of FACS buffer (if using 96-well plate add 200 μl),
and centrifuge at 650 G for 2 min, at 4°C.
3.3.2 Index-sorting of human DC 8. Aspirate the supernatant.
9. Optional: If you chose to include CADM1 into your staining
For detailed preparation of single-cell suspension from human tis- (to target cDC1) and since a purified antibody is used to stain
sues we refer to “Chapter IX Mononuclear phagocyte phenotypes” for CADM1 on DCs, you will need to perform an additional
in Cossarizza et al. (2021) [27]. The tissue processing protocols staining step (otherwise proceed with step 14.):
described in Cossarizza et al. (2021) [27] are perfectly suitable a. Re-suspend the cell pellet in 50 μl of FACS buffer contain-
for processing of human DC for flow cytometry, as well as for scR- ing anti-Chicken-IgY-Alexa-Fluor 647 (to target CADM1).
NAseq. Incubate for 15 min, at 4°C. Then add 2 ml of FACS buffer
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Eur. J. Immunol. 2023;53:2249925 Single cell RNA sequencing of human tissue DC 21 of 96
(if using 96-well plate add 200 μl) and centrifuge at 650 to analysing their own data (https://satijalab.org/seurat/articles/
G for 2 min, at 4°C. pbmc3k_tutorial.html).
b. Aspirate the supernatant.
10. Re-suspend the cell pellet in 200–400 μl of FACS buffer, fil-
ter through a 70 μm cell strainer into a new (clean) 5 ml 3.4.1 Pre-processing, quality assessment and control, and
polystyrene FACS tube, and analyze using a suitable flow analysis of SMARTseq2 single-cell transcriptome data
cytometer.
1. Align paired-end raw reads to the human reference genome
For indexed-sorting, cells were recorded and sorted using (GRCh38 version 25 release; Gencode) using the RSEM pro-
an ARIAIII 5L (70 μm nozzle; BD Biosciences). Fcs files were gram version 1.3.0. A step-by-step tutorial for RSEM is avail-
exported and analyzed using FlowJo v10.8.1 (BD Biosciences). able on https://github.com/bli25/RSEM_tutorial [28]. Fol-
lowing the tutorial calculate the “Transcript Per Million read”
(TPM) values. These TPM values are then used for down-
3.3.3 Generation of SMARTseq2 single-cell transcriptome stream analysis.
data 2. Next, using the Seurat R package (following the “Seurat –
Guided Tutorial – 2.700 PBMCs” tutorial) create a Seurat
Independent of the sequencing method chosen, the basic pro- object first, by using the in step 1 generated TPM as input.
cessing steps are shared across platforms and include 1. cell 3. Run the quality control (QC). QC metrics are then visualized
isolation, followed by 2. cell lysis to access the mRNA within, as violin plots. QC has to be assessed individually for each data
3. the conversion of mRNA molecules to cDNA, while barcod- set. If all cells have passed the QC continue with step 4, other-
ing each molecule with a unique identifier (UMI; unique cell wise remove cells that failed the QC before moving on.
and molecular barcode), and last 4. pooling of barcoded/labeled 4. Normalize and scale data using the ‘NormalizeData()’ and
molecules, library construction, and sequencing. Here, we provide ‘ScaleData()’ function from Seurat.
an example generating index-sorted SMARTseq2 transcriptome 5. As a next step, highly variable genes can be identified and plot-
data. ted (for cells that passed the QC) using the ‘FindVariableFea-
tures’ and ‘VariableFeaturePlot’ functions from Seurat, respec-
1. Index-sort the above prepared and stained cells directly into tively.
96-well plates containing 3 μl of lysis buffer (0.2% Triton X- 6. Perform the principal component analysis (PCA) using the
100, Sigma-Aldrich), using a 70 μm nozzle (BD FACS ARIAIII highly variable genes identified in 3.4.1.5.
5L (BD Biosciences).
2. Prepare single cell cDNA library following the SMARTSeq v2 Note: All scRNAseq dot plots and meaning plots displaying the
protocol [26], with a few modifications: gene expression levels or mean signature genes can be generated
a. Use Lysis buffer (as above). using R or SeqGeq v1.6 (Flow Jo LLC).
b. Use 200 pg cDNA with 1/5 reaction of Illumina Nextera XT
kit (Illumina, San Diego, CA, USA).
c. The length distribution of the cDNA libraries can be 3.4.2 Dimensionality reduction
monitored using a DNA High Sensitivity Reagent Kit on
the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, A Uniform Manifold Approximation and Projection (UMAP) [29]
USA). of the scRNAseq data will allow us to visualize and explore the
3. All samples are subjected to an indexed paired-end sequenc- datasets in a dimensionally reduced space:
ing run of 2×151 cycles on an Illumina HiSeq 4000 sys-
tem (Illumina, San Diego, CA, USA), with 300 samples/ 1. To generate such a UMAP use the RunUMAP() function in Seu-
lane. rat using the significant PCs identified previously (in step 6
of 3.4.1 Pre-processing, quality assessment, and control and
analysis of SMARTseq2 single-cell transcriptome data), based
3.4 Data analysis on elbow/scree plots or Jackstraw obtained from the Seurat
analysis.
Indexed-flow cytometry data can be analyzed in a similar man- 2. Seurat KNN clustering is implemented for scRNAseq data for
ner as classical flow cytometry data using FlowJo (BD). For more an unsupervised classification of cells types (Fig. 6A).
detailed analyses and for combining the index-information with
the SMARTSeq2 sequencing data the following pipeline can be Note: To determine an optimal number of clusters for down-
used. Most of the analyses are performed using R and the below- stream analyses, multiple cluster resolutions are used. Here, Clus-
mentioned R packages. These should be installed prior analyses. tree Rpackage (https://github.com/lazappi/clustree) is then used
For first-time users, we recommend practicing with the “Seurat – to visualize clusters at different resolutions when interrogating
Guided Tutorial – 2.700 PBMCs” provided by the Satija lab prior clustering.
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22 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Figure 6. Analyses of indexed-SMARTseq2 scRNAseq data of human tissue DC. (A) Heatmap of differentially expressed genes (DEG) in between
SNN clusters (obtained using the Seurat pipeline) of indexed-SMARTseq2 (SS2) scRNAseq data. The relative frequency in each cluster of tissues and
cell type annotation is annotated (obtained from DEG and from indexed-data protein expression analysis, not shown). (B) KNN clusters (left panel),
cell subsets identification based on indexed-data protein expression analysis (middle panel) and of tissue of origin (right panel) are overlayed
onto the UMAP space. (C) Meaning plots showing the relative expression of “indexed” protein markers included in the indexed-SS2 data. (Figure
reproduced with permission from Mulder et al., 2021 [30]).
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Eur. J. Immunol. 2023;53:2249925 Single cell RNA sequencing of human tissue DC 23 of 96
3.4.3 Defining clusters 3.4.5 Integrating indexed-flow cytometry data with
scRNAseq data
Differentially expressed gene (DEG) analysis is performed to aid
cluster annotation. Canonical markers arising from the DEG anal- Extract the mean fluorescence intensity of the analyzed indexed-
ysis will allow for matching the unsupervised clustering to known flow cytometry from FlowJo. Add these data to the metadata of
cell types on a transcriptional level (Fig. 6B). the Seurat object according to the cell IDs. This allows for the
integration of protein information with the scRNAseq analysis for
1. Calculate DEGs using normalized values with a logFC thresh- comparative analyses by overlaying protein expression directly
old of 0.25. Perform a likelihood-ratio test for single-cell gene onto the UMAP, that was generated using transcriptomic analysis
expression (bimodal test) prior to correction for multiple test- (Fig. 6C). This can be done following the steps detailed in 3.4.4.
ing, using the Bonferroni method.
2. Generate and plot a heatmap to visualize e.g., the top 20 DEGs
(or all DEGs if these are less than 20) for each cluster. 3.5 Pitfalls
3. If available, additional metadata (such as tissue identity, DC
populations identified using flow cytometry gating of the pro- Beyond transcriptomic variations in between cells obtained from
tein data, etc.) can be overlayed onto the heatmap to ensure different patients/donors or different tissues (which correspond
robustness of the cell type identity assigned to each cluster to biological variations due to patient-to-patient differences and
here (Fig. 6B). to different transcriptomic imprinting linked to the variation in
tissue microenvironmental signals), a strong bias of scRNAseq
Note: In some cases, closely related clusters could be grouped data can be due to technical variation in between data obtained in
together to form mega-clusters with biological relevance. For different batches. To circumvent this technical bias, several algo-
this, the average expression of each gene obtained from the rithms (Seurat V4, Harmony, LIGER, and several others) allow to
DEG analysis for each Phenograph cluster is derived, to per- integrated datasets obtained in different batches or using different
form a Spearman correlation. A dendrogram heatmap can scRNAseq techniques in a single UMAP or tSNE space.
then be generated using the Ward’s method for hierarchi- Depending on the technique employed, the number of tran-
cal cluster analysis in the pheatmap Rpackage (https://cran.r- scripts sequenced for each cell varies from 30,000 reads (droplet-
project.org/web/packages/pheatmap/index.html). based methods such as 10× Genomics Chromium or BD Rhap-
sody) to up to 1 million reads (SMARTseq2). This only allows to
sequence a fraction of the total mRNA pool in each cell and thus,
3.4.4 Plotting the expression of genes or proteins of interests does not allow to detect low copy number transcripts. Further-
more, due to the short half-life of a transcript (much shorter than
Selected genes of interest or mean signature genes can be plotted that of proteins), cells that might have expressed a transcript a
as meaning plots to display the gene expression levels directly on few hours before their analysis might not express it anymore at
the UMAP space (for global visualization) or as violin plots (for the time of analysis. This phenomenon is called “zero inflation”
comparison across different metadata groups/clusters). This can that can thus be due to biological or technical reasons.
be generated using R or SeqGeq v1.6 (Flow Jo LLC). Most immune cell subsets have been identified using protein
markers quantified by flow cytometry. With the advent of RNAseq,
1. Using R: researchers have observed that there can be a discrepancy in
a. Visualize individual gene/protein expressions using the between RNA and protein expression. For example, CD4 is most
FeaturePlot() or VlnPlot() function. highly expressed by CD4+ T cells at the protein level, while the
b. Calculate a module score using the AddModuleScore func- expression of CD4 mRNA is the highest on mononuclear myeloid
tion. Use this to visualize the mean expression of a set of cells such as monocyte, macrophages, and certain subsets of DC.
gene signatures using FeaturePlot() or VlnPlot().
2. Using SeqGeq,
a. Similar to standard flow cytometry data analyses in FlowJo, 3.6 Top tricks
individual protein expression levels can be visualized using
the color axis function. The viability of cells and the time spent from tissue harvesting
b. To plot mean signatures, select <Genes> → <New Static to cell processing to generate the RNA library is critical to avoid
Gene Stat> technique-related gene expression variations. Human tissue sam-
i. Select genes (e.g., XCR1, CADM1, WDFY4) and name ples should be processed as soon as possible upon reception. Leav-
this list “cDC1 genes” ing samples overnight prior to processing is not recommended.
ii. On the workspace under <Operations>, select Ensure that incubation times are strictly followed as per the pro-
<Mean> and <Apply operations> tocols. Due to the delicate nature of RNA, the workstation should
iii. The mean signature of the list can now be visualized be RNAse free and personal protective equipment (e.g., lab coat,
with the color axis function as “cDC1 genes <Mean>” gloves, mask) should be worn when processing the samples. We
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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24 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
suggest to work quickly but precisely, keeping cells and RNA Here, we present a protocol combining FACS isolation of
chilled at all times possible. splenic cDC subsets with MS-based proteomic analysis. The pro-
tocol is optimized for sample types where cell numbers are lim-
ited and require 10,000–30,000 cells as input material, corre-
sponding to between one and five micrograms of total protein,
4 FACS isolation and mass depending on DC subtype and maturation state. To keep purifi-
spectrometry-based proteomic analysis of cation steps at a minimum and avoid sample loss, pure PBS is
rare mouse DC populations used as sheath buffer, CD11c+ splenocytes are washed with PBS
prior to FACS analysis to remove FCS and are directly sorted into
4.1 Introduction a sodium dodecyl sulfate (SDS)-containing lysis buffer. The work-
flow is compatible with single-pot, solid-phase-enhanced sample
Acting at the interface of innate and adaptive immunity, den- preparation (SP3) for proteomic analysis [41–43]. The presented
dritic cells (DC) fulfill a multitude of crucial functions in orches- protocol is devised and particularly attractive for the bulk char-
trating and initializing pathogen-specific immune responses. DC acterization of cell-specific proteomes of rare, low abundant cell
can be grouped into two major classes, natural type I interferon- types.
producing plasmacytoid DC (pDC) and conventional (c)DC. The
latter can be further divided into phenotypically and functionally
distinct subpopulations. While cDC1 (including splenic XCR1+ 4.2 Materials
CD8α+ cDC1 and their CD103+ counterparts in peripheral tis-
sues) are specialized in antigen (Ag) cross-presentation, CD8+ 4.2.1 Reagents
T cell activation and participate in antiviral immunity, cDC2
(SIRPα+CD11b+) are thought to have a predominant role in All antibodies used in the present protocol are listed in Table 11.
CD4+ T cell priming and immune responses against extracellu- All necessary reagents and chemicals are listed in Table 12.
lar pathogens. Importantly, cDC1 and cDC2 subpopulations can be
further delineated into several smaller and less-well characterized 4.2.1.1 Buffers for single-cell preparation and FACS analysis.
tissue-specific subsets that exhibit diverse immune-modulatory FCS: Quickly thaw FCS at 37°C in a water bath. Once completely
functions. For example, splenic cDC2 consist of at least two pre- thawed, incubate for 60 minutes at 56°C in the water bath to
dominant lineages, called cDC2A and cDC2B [31]. While cDC2A destroy complement activity. Directly aliquot the warm FCS into
are characterized by high ESAM expression, cDC2B are ESAMlo 50 mL portions and store at –20°C. Avoid freeze-thaw cycles. Use
and selectively express Clec12a [31–33]. ESAMhi cDC2A are effi- aseptic techniques during the whole procedure.
cient in MHC-II Ag-presentation and CD4+ T cell activation, but
poor in the production of pro-inflammatory cytokines, which is a DNAse I: Dissolve Deoxyribonuclease I (DNAse I) in PBS to reach
characteristic of ESAMlo cDC2B [31–34]. a final concentration of 500 U/mL. Prepare aliquots, store dis-
Cell surface markers like ESAM, XRC1, and SIRPα can be solved DNAse I at –20°C, and avoid freeze-thaw cycles. Use sterile
used to analyze distinct splenic cDC populations by fluorescence- solutions and aseptic techniques.
activated cell sorting (FACS). FACS is widely used for clinical
applications and in life sciences due to its ability to isolate distinct Collagenase IV: Dissolve Collagenase IV in PBS to reach a con-
cell populations with high purity including even rare subpopu- centration of 10,000 U/mL. Prepare aliquots, store dissolved Col-
lations [35, 36]. Subsequent proteomic analysis of isolated cells lagenase IV at –20°C, and avoid freeze-thaw cycles. Use sterile
by liquid chromatography–mass spectrometry (LC–MS) allows to solutions and aseptic techniques.
further unravel cell-specific functionalities. MS is unparalleled
in its ability to characterize and quantify proteins in an unbi- Collagenase IV/DNAse I Digestion mix: Per mouse, prepare
ased manner at the molecular level [37, 38]. By interfacing both 1 mL of digestion solution containing 200 U/mL Collagenase IV
techniques, FACS, and MS, it is possible to determine the cell- and 0.5 U/mL DNAse I in RPMI medium.
type-specific protein repertoire of distinct cellular subpopulations
to decode their cell-type-specific proteomes and functionalities FACS buffer: Phosphate buffered saline solution (PBS) containing
[39–41]. However, the proteomic analysis of a limited number 3% FCS and 2 mM EDTA.
of FACS-sorted cells can be very challenging. Often, substances
used in the FACS sheath fluid including salts, polyethylene glycol PBS wash buffer. Dissolve PBS powder (Roti®-PreMix PBS)
(PEG), or other viscosity-altering molecules and/or components according to manufacturer’s instruction in LC–MS grade water.
of FACS sample buffers (e.g. albumin, fetal calf serum (FCS)) can Can be stored at 4°C for up to 1 month and also be used for the
impair LC-MS downstream analysis. Hence, additional purifica- preparation of the SDS lysis buffer.
tion steps may be required prior to proteolytic digestion [40, 41]
which can lead to sample loss and irreproducible results, particu- SDS lysis buffer (5×). Dissolve 9.53 mg DDT and 25 mg SDS
larly when handling low amounts of input material. in 2.25 mL LC-MS grade water, add 250 μL of PIC stock (50x)
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 FACS isolation and mass spectrometry-based proteomic analysis of rare mouse DC populations 25 of 96
Table 11. Antibodies for FACS analysis
Monoclonal antibodies Clone Conjugate Isotype Manufacturer cat. no.
Anti-CD11c N418 FITC Hamster IgG2 eBioscience 11.0114
Anti-CD172a (anti-SIRPα) P84 PerCP-eFl710 Rat IgG1,κ BD Biosciences 46-1721
Anti-ESAM 1G8/ESAM PE Rat IgG2a,κ Biolegend 136203
Anti-I-A/I-E (anti-MHC-II) M5/114.15.2 APC Rat IgG2b,κ Biolegend 107618
Anti-XCR1 ZET BV510 Mouse IgG2b,κ Biolegend 148218
Anti-CD16/32 (Anti-FcyRIIB/III) 2.4G2 Purified Rat IgG2a,λ Biolegend 101302
to achieve a final concentration of 25 mM DTT, 10% (w/v) SDS 4.2.1.2 Buffers and solutions for proteomic sample preparation
and 5× PIC concentration. Instead of water, PBS can be used as (SP3 digest).
well. Prepare buffer freshly before use. Note: Buffering capacity is SP3 bead stock solution (20 µg solids/µL). Combine 20 μL of
provided by PBS from the sorting buffer. Sera-Mag carboxylate-modified magnetic particles (hydrophobic,
Table 12. Reagents and chemicals used in the present protocol
Reagent/Chemical Manufacturer cat. no.
Single-cell preparation and FACS analysis
ACK Lysing Buffer Thermo Fisher Scientific Inc. A1049201
Anti-CD11c Microbeads UltraPure, Hamster IgG2 Miltenyi Biotec 130-108-338
Collagenase Type IV Worthington LS0004186
cOmplete Protease Inhibitor Cocktail (PIC) Roche 1697498001
Deoxyribonuclease I (DNAse I) Roche 10104159001
1.4-Dithiothreitol 99%, p.a. (DTT) Carl Roth 6908.1
Dulbecco´s Phosphate Buffered Saline (PBS) w/o calcium and Merck KGaA D8537
magnesium
Ethylenediaminetetraacetic acid (EDTA) Merck KGaA E5134-500G
Fetal Bovine Serum (FBS) Gibco 10270-106
Fixable Viability Stain (L/D), FVS780-conjugated BD Biosciences 565388
Roswell Park Memorial Institute (RPMI) 1640 Medium Thermo Fisher Scientific Inc. 11530586
Roti®-PreMix PBS Carl Roth 0890.1
Sodium dodecyl sulfate (SDS) AppliChem A7249-0250
Water Rotisolv®, Ultra LC-MS Carl Roth HN43.1
Proteomic sample preparation (SP3 digest)
Absolute ethanol Merck KGaA 34852-M
Acetic acid Carl Roth 3738.2
Acetonitrile (ACN), Rotisolv®, Ultra LC-MS Carl Roth HN40.1
Ammonium bicarbonate Carl Roth T871.1
1.4-Dithiothreitol 99%, p.a. (DTT) Carl Roth 6908.1
Dimethylsulfoxide (DMSO) Carl Roth HN47.1
Formic acid (FA), ROTIPURAN®, LC-MS Grade Carl Roth 1EHK.1
Iodoacetamide BioUltra (IAA) Merck KGaA I1149-25G
Sera-MagTM Magnetic carboxylate modified particles Merck GE24152105050250
(hydrophilic)
Sera-MagTM magnetic carboxylate modified particles Merck GE44152105050250
(hydrophobic)
Trypsin Gold, MS Grade Promega V5280
Water Rotisolv®, Ultra LC-MS Carl Roth HN43.1
LC-MS analysis
Acetonitrile (ACN), Rotisolv®, Ultra LC-MS Carl Roth HN40.1
Formic acid (FA), ROTIPURAN®, LC-MS Grade Carl Roth 1EHK.1
Isopropanol (2-Propanol) Rotisolv®, Ultra LC-MS Carl Roth 0733.1
Sodium hydroxide Carl Roth 6771.2
Water Rotisolv®, Ultra LC-MS Carl Roth HN43.1
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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26 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
provided at a concentration of 50 μg/μL by the vendor) and Calibration solution, 10 mM sodium formate. To prepare a
20 μL of Sera-Mag Speed-Bead carboxylate-modified magnetic 10 mM sodium formate calibration solution, mix 12.5 mL water,
particles (hydrophilic, 50 μg/μL) in a 1.5 mL-tube. Add 160 μL 12.5 mL isopropanol, 50 μL formic acid, and 250 μL sodium
of water and mix. Place the tube with beads on a magnetic rack hydroxide.
and remove supernatant after beads have settled. Off the rack, Equipment required for the present protocol is listed in
add 200 μL of water and mix. Repeat wash steps two further Table 13.
times. Recover beads in 100 μL of water. SP3 bead stock can be
stored at 4°C for up to 1 month.
4.3 Step-by-step sample preparation
1.4-Dithiothreitol (DTT) stock solution (200 mM). Weigh 3–
5 mg of DTT directly into a fresh 1.5-mL tube and dissolve in 4.3.1 Single-cell preparation for FACS analysis
water/or PBS to a final concentration of 200 mM. As DDT is
oxygen-sensitive DDT stock solutions and DTT-containing buffers 1. Euthanize mice using CO2.
should be prepared freshly before use. 2. Dissect out the spleen.
3. Cut tissue in grain size pieces and incubate in 1 mL digestion
Iodoacetamide (IAA) stock solution (400 mM). Weigh 10– mix while shaking at 37°C for 30 min.
20 mg of IAA directly into a fresh 1.5-mL tube and dissolve in 4. Add EDTA to a 10 mM final concentration and incubate the
water/or PBS to a final concentration of 400 mM. As IAA is light- cell suspension for 5 min at 4°C.
sensitive IAA stock solutions and IAA-containing buffers should be 5. Add cell suspension onto a 40 μm cell strainer (placed on a
prepared freshly before use and kept in the dark. 50 mL tube).
6. Wash cell strainer by adding 10 mL FACS buffer.
70% (v/v) ethanol (EtOH). Mix 70 mL of absolute EtOH with 7. Centrifuge cells for 5 min at 400 × g at 4°C. Aspirate super-
30 mL of water. natant completely.
8. Resuspend cell pellet in 1 mL ACK lysis buffer to lyse red
Ammonium bicarbonate buffer (50 mM). Weigh 40 mg of blood cells. Incubate at room temperature for 5 min.
ammonium bicarbonate and transfer to a 15 mL-polypropylene 9. Add 10 mL of FACS buffer.
tube. Dissolve in 10 mL of water to a final concentration of 10. Centrifuge cells for 5 min at 400 x g at 4°C. Aspirate super-
50 mM. Prepare freshly prior to use. natant completely.
11. Resuspend cell pellet in 400 μL FACS buffer.
Trypsin stock solution (1 µg/µL). Dissolve lyophilized trypsin in 12. Commercially available, purified rat anti-mouse anti-
50 mM acetic acid prepared in water to a final concentration of CD16/32 is employed to block the Fc-gamma RIIB/III recep-
1 μg/μL. Store 5 μL-aliquots at –80°C. tors, thereby avoiding unspecific recognition of staining anti-
bodies by cell-bound Fc-gamma receptors. For this, calculate
Trypsin digestion buffer. Dilute trypsin stock solution in ammo- the amount of Fc-block and prepare in 25 μL FACS buffer per
nium bicarbonate buffer. For proteolytic digestion, trypsin-to- well.
protein ratio should be approx. 1:25 (w/w) and the final working 13. Apply the 25 μL Fc solution onto cells, mix well and incubate
volume 5 μL. Prepare freshly before use. for 5 min at 4°C.
14. To isolate CD11c+ dendritic cells from the single-cell suspen-
Peptide elution solution, 2% (v/v) DMSO. Mix 98 μL of water sion, incubate cells from one spleen with 100 μL anti-mouse
with 2 μL of DMSO. Prepare freshly before use. CD11c MACS MicroBeads UltraPure. Perform MACS purifica-
tion and isolation according to the manufacturer’s protocol.
15. Elute the CD11c+ cDC from the LS MACS column by apply-
4.2.1.3 Buffers and solutions for LC-MS analysis. ing 5 mL of FACS buffer to the column, followed by firmly
Solvent A. 0.1% (v/v) FA in water. Degas solvent, either by sparg- pushing the plunger into the column.
ing with helium or sonication for at least 15 min. 16. Count isolated cells and continue with section 4.3.2.
Solvent B. 0.1% (v/v) FA in ACN. Degas solvent, either by sparg-
ing with helium or sonication for at least 15 min. 4.3.2 Flow cytometric purification, collection and lysis of
splenic cDC subsets
Needle and fluidics wash. Wash solution A: 1% (v/v) FA in water.
Wash solution B: 0.1% (v/v) FA in ACN. Degas solvents, either by 1. Prior to FACS analysis, aliquot SDS lysis buffer (5×) in fresh
sparging with helium or sonication for at least 15 min. 1.5 mL protein LoBind Eppendorf tubes. Note: Depending
on the nozzle and sorting mode, calculate the volumes of
Transport liquid. 0.1% FA (v/v) in water. Prepare freshly (i.e. 10% (w/v) SDS lysis buffer (5×) required to achieve a final
once a week). concentration of 2% (w/v) SDS, 5 mM DTT, and 1× PIC
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Eur. J. Immunol. 2023;53:2249925 FACS isolation and mass spectrometry-based proteomic analysis of rare mouse DC populations 27 of 96
Table 13. Summary of required equipment
Equipment Manufacturer cat. no.
Single-cell preparation and FACS analysis
2 mL microcentrifuge tubes Sarstedt AG 72.695.200
15 mL tubes Greiner bio-one 188271 or equivalent
50 mL tubes Greiner bio-one 227261 or equivalent
BD FACSAria I BD Biosciences
Centrifuge “Z 446 K” Hermle LaborTechnik
FACS tube Corning 352008
Falcon® 40μm cell strainer Corning 352340
MACS LS Columns Miltenyi Biotec 130-042-401
Neubauer chamber 0.100 mm; 0.0025 mm2 Superior Marienfeld
PipetBoy Fisher Scientific 11701258 or equivalent
PipetMan (P10-P1000) Gilson
Pipette tips Brand
Protein LoBind 1.5 mL Eppendorf tubes Eppendorf 0030108116
QuadroMACS Separator Miltenyi Biotec
Serological pipettes (1-25 mL) Gilson
Proteomic sample preparation (SP3 digest)
Bioruptor Plus device Diagenode B01020001 or equivalent
Benchtop centrifuge holding 1.5-mL tubes Thermo Scientific 10406173 or equivalent
(up to 21,000 x g)
Magnetic rack(s) holding 1.5-mL microfuge tubes Cell Signaling 7017S or equivalent
Protein LoBind 1.5 mL Eppendorf tubes Eppendorf 0030108116
Protein low binding pipet tips Corning TF-20-L-R-S,
(Axygen®Maxymum Recovery®Universal Fit Pipet Tips) TR-222-C-L-R,
TF-1000-L-R-S
ThermoMixer equipped with a 1.5-mL tube holder Grant Instruments 97011-930 or equivalent
LC-MS analysis
Aurora Series UHPLC column + CaptiveSpray insert Ionopticks AUR2-25075C18A-CSI
CaptiveSpray nanoBooster Source Bruker Corporation 1820410 or equivalent
Column toaster Bruker Corporation 1862996 or equivalent
NalgeneTM syringe filter Thermo Scientific 722-2000
Syringe filter, PTFE, with active charcoal reservoir Sartorius 17840———-Q
nanoElute® LC system Bruker Corporation 1832734 or equivalent
timsTOF Pro (2) Bruker Corporation 1900000 or equivalent
Total recovery glass vials Waters Corporation 600000750cv
after the cell sorting; e.g. 22.5 μL of 10% (w/v) SDS lysis 7. Wash cells by adding 2 mL PBS prepared with LC-MS grade
(5×) buffer are needed for the lysis of 30,000 cells when water, resuspend the cells and spin down for 5 min at 400 x g
using a 100 μm nozzle and 4-way purity mode. According at 4°C. Aspirate supernatant completely. Repeat this washing
to the manufacturer droplet size per cell corresponds to 3 nL step.
when using these settings resulting in a total “sort” volume 8. Resuspend cells at a concentration of 1×107 cells/mL in PBS
of 90 μL. (prepared with LC-MS grade water). Note: Do not use volumes
2. Calculate and prepare the antibody master mix. Note: Don’t below 500 μL for resuspension of cells.
forget to include controls and optional extra staining for instru- 9. Sort cDC subsets by gating on high expression of CD11c and
ment setup. MHCII and the absence or presence of SIRPα, ESAM, and
3. Centrifuge cells for 5 min at 400 x g at 4°C. Aspirate super- XCR1. Sort 30,000 cells directly into a protein LoBind Eppen-
natant completely. dorf tube containing 5× SDS-lysis buffer (see 3.2. step 1). To
4. Apply 200 μL antibody master mix onto cells, mix well by sort cDC subpopulations, we used a nozzle size of 100 μm,
pipetting up and down and incubate for 30 min at 4°C in the and “4-way purity” sort mode, which required 22.5 μL of 5×
dark (fridge). SDS-lysis buffer.
5. Fill tubes with 5 mL FACS buffer. 10. Incubate samples directly after FACS isolation at 95°C for
6. Centrifuge cells for 5 min at 400 x g at 4°C. Aspirate super- 5 min to promote cell disruption and protein solubilization.
natant completely. 11. Cool down samples for 5 min on ice.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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28 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
12. Place samples into the Bioruptor device and sonicate at 4°C peptide binding to the beads, add ACN to a final concentra-
at maximum intensity applying in total 15 cycles (with 30 s tion of 95% (v/v). Immediately vortex-mix the samples.
on/off intervals each) to further promote cell lysis and to 11. Incubate the samples for 20 min at room temperature. Briefly
shear chromatin. vortex-mix the samples during the first 10 min as soon as sed-
13. Process samples by single-pot solid-phase-enhanced sam- imentation of the peptide-bead aggregates is observed. After-
ple preparation (SP3, see section 3.4.). Pause point: cellular ward, allow the beads to settle.
lysates can be also stored at –80°C until further processing. 12. Place the samples into a magnetic rack and capture the beads
at the tube wall. Wait at least 2 min until all beads are
migrated to the tube wall. Remove and discard the super-
4.3.3 Proteolytic digestion using single-pot natant. Note: To increase the peptide recovery, the supernatant
solid-phase-enhanced sample preparation (SP3) can be incubated a second time with SP3 beads [41] (see also
section 5). Continue with step 11.
1. Reduce disulfide bonds by adding DTT stock solution to the 13. Rinse the pelleted SP3 beads on the magnetic rack by adding
samples resulting in a final concentration of 10 mM DDT (e.g. and carefully pipetting up and down 200 μL of ACN. Remove
0.5 μL of DTT stock solution per 10 μL of lysate). Incubate and discard the supernatant. Transfer the tubes with opened
samples at 45°C for 30 min. lid from the magnetic rack and let residual ACN evaporate.
2. To alkylate free cysteine residues, add IAA stock solution to 14. Reconstitute the beads in 10 μL of 2% (v/v) DMSO to elute
achieve a final concentration of 40 mM (e.g. 1 μL per 10 μL purified peptides from the SP3 beads. Incubate the samples
of lysate). Incubate samples in the dark for 30 min at room in an ultrasonic bath for 1 min to improve peptide recovery.
temperature. Note: If peptides were recovered a second time with SP3 beads
3. To quench the alkylation reaction, add 0.75 μL of DTT solu- (see “Note” step 12), combine both SP3 beads in a total of 10
tion per 10 μL of lysate resulting in a final concentration of μL 2% (v/v) DMSO.
15 mM DDT. 15. Centrifuge the tubes for 2 min at 12,500 x g and place them
4. Shortly vortex the SP3 bead stock solution. Add 2 μL of the onto the magnetic rack. Recover the supernatant containing
bead stock to each sample. Shortly vortex-mix the suspension tryptic peptides after all beads have settled on the tube wall.
and avoid excessive pipetting to prevent sample loss. Note: If Make sure not to recover any beads.
the sample volume exceeds 50 μL, adjust the amount of beads 16. To proceed with LC-MS analysis, mix samples with diluted
that are added to achieve a concentration of at least 0.5 μg/μL FA to a final concentration of 0.1% (v/v) FA. If you opt for
of SP3 beads in the final bead-sample suspension. a final sample volume of 20 μL, for example, add 10 μL of
5. Add ACN to a final concentration of 70% (v/v) to induce 0.2% (v/v) FA to the sample.
aggregation and binding of proteins to the SP3 beads. Vortex-
mix the samples immediately.
6. Incubate the samples for 20 min at room temperature. Briefly 4.3.4 Liquid chromatography-mass spectrometry (LC-MS)
vortex-mix the samples during the first 10 min as soon as sed-
imentation of the protein-bead aggregates is observed. After- Note: In the present study, samples were analyzed on a tim-
ward, allow the beads to settle. sTOF Pro 2 mass spectrometer coupled to a nanoElute LC sys-
7. Place the samples into a magnetic rack to capture the beads at tem (Bruker Corporation) using ion mobility-enhanced data-
the tube wall. Wait at least 2 min until all beads are migrated independent acquisition (DIA-PASEF) as described by Meier et al.
to the tube wall. Remove and discard the supernatant. [44] However, mass spectrometric analysis of the samples can be
8. Rinse the pelleted SP3 beads on the magnetic rack by adding conducted on any high-resolution, state-of-the-art LC-MS instru-
and carefully pipetting up and down 200 μL of 70% (v/v) ment platform.
EtOH. Remove and discard the EtOH-containing supernatant.
Repeat this step a second time with 70% (v/v) EtOH and 1. Transfer samples into glass vials and place them into the
once with ACN. Transfer the tubes with opened lid from the autosampler of your LC system. In the present study, we used
magnetic rack and let residual ACN evaporate. a nanoElute LC platform for the separation of tryptic peptides.
9. Add 5 μL of trypsin digestion buffer and resuspend beads. 2. Mount the CaptiveSpray source on the timsTOF Pro 2 mass
Incubate the samples at 37°C overnight to promote tryp- spectrometer.
tic digestion of proteins. Note: We typically use a trypsin-to- 3. Install a system comprising reversed-phase C18 column (e.g.
sample ratio of 1:25 (w/w).To estimate the protein content in Aurora UHPLC emitter column, 25 cm x 75 μm 1.6 μm,
dendritic cells, we used DC 2.4 cells as reference, which contain IonOpticks).
around 80–90 pg of protein per cell corresponding to approx. 4. Perform mass and tims calibration according to the manufac-
2.4-2.7 μg per 30,000 cells. Hence, 5 μL of digestion buffer turer’s protocol.
should contain around 108 ng of trypsin. 5. To setup your chromatographic method, choose “one-column
10. After tryptic digestion, resuspend the beads by shortly vor- separation” mode in the chromatographic method editor, set
texing the tubes. To purify the peptide mixture and promote the column temperature to 50°C and choose default settings
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 FACS isolation and mass spectrometry-based proteomic analysis of rare mouse DC populations 29 of 96
Figure 7. Proteomic workflow and gating strategy for the analysis of cDC subsets. A) Graphical illustration of the presented proteomic protocol combing
FACS isolation and LC-MS downstream analysis of splenic cDC subsets. (B) Flow cytometry gating strategy for the isolation of splenic cDC subsets.
Representative cytometry plots of the gating strategy used for sorting XCR1+SIRPα– cDC1 and XCR1–SIRPα+ cDC2 from the mouse spleen. CD11c
MACS-enriched splenocytes from 10 to 16-week-old mice were stained with the indicated antibodies, and subsequently, cDC were defined as viable
gated CD11c+MHC-II+ cells. cDC subsets were based on XCR1 and SIRPα, where the expression or absence of ESAM further defined cDC2 as cDC2A
or cDC2B populations, respectively [31, 33, 45].
for separation column equilibration and sample loading Note: 4.4 Data analysis
We set the column equilibration volume to 5.0 instead of 4.0.
Additionally, we lowered equilibration and sample loading pres- Here, we describe a detailed protocol for the MS-based proteomic
sure to 600 bar to increase column life time. characterization of mouse splenic cDC1 and cDC2 subsets isolated
6. Program the following gradient for peptide separation in the by FACS (Fig. 7A). The detailed gating strategy for the selection
method editor: Increase solvent B from 2% (v/v) to 25% (v/v) of cDC1 (CD11c+MHC-II+ XCR1+SIRPα–), cDC2A (CD11c+MHC-
in 34.5 min, then to 37% (v/v) over 3.8 min, followed by II+ SIRPα+ESAM+XCR1–) as well as cDC2B (CD11c+MHC-II+
ramping up B% to 95% (v/v) over another 3.8 min. Wash col- SIRPα+ESAM–XCR1–) cells is summarized in Fig. 7B. Cells were
umn for 5 min with 95% (v/v) B. Set the flow rate to 400 directly sorted into SDS-lysis buffer and processed for LC-MS anal-
nL/min. Note: We would recommend to adjust gradient accord- ysis by SP3 to minimize sample loss. The exemplary dataset pro-
ing to column and sample type. vided with this protocol contains five biological replicates of each
7. Analyze eluting peptides in positive mode ESI-MS on a cDC subpopulation. LC-MS raw data processing, peptide identi-
timsTOF Pro 2 instrument. We recommend to analyze the fication, and label-free quantification (LFQ) analysis of proteins
samples using parallel accumulation serial fragmentation were performed using DIA-NN (v1.8) [46]. To this end, samples
(PASEF)-enhanced data-independent acquisition mode (DIA) were processed using library-free mode with standard parameters
as described in detail by Meier et al. [44] Operate the dual including some slight modifications as summarized in Table 14.
TIMS (trapped ion mobility spectrometer) at a fixed duty The required FASTA protein database containing 17,068 reviewed
cycle close to 100% using equal accumulation and ramp times protein entries of mouse and 172 common contaminant proteins
of 100 ms each spanning a mobility range from 1/K0 = was obtained on April 16, 2021, from uniprot.org.
0.6 Vs cm−2 to 1.6 Vs cm−2. Define 36× 25Th isolation win- To identify differentially regulated proteins in the different
dows from m/z 300 to 1,165 (i.e. fifteen diaPASEF scans per cDC subsets, further statistical analysis was conducted using
acquisition cycle). Ramp the collision energy linearly as a func- the protein level LFQ values reported by DIA-NN. A two-sided
tion of the mobility from 59 eV at 1/K0 = 1.3 Vs cm−2 to 20 eV t-test assuming equal variances was performed considering only
at 1/K0 = 0.85 Vs cm−2. proteins that had been identified in at least three biological
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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30 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 14. Settings used for data processing in DIA-NN cytosolic proteins as well as transcription factors (see Table 15).
Gene ontology analysis revealed an enrichment of proteins asso-
Parameter Setting used
ciated with antigen processing and presentation via MHC class
Precursor ion generation I in cDC1 cells, whereas proteins identified in cDC2 cells are
FASTA digest for library-free search selected mainly involved in proinflammatory processes (e.g. regulation
library-free of interferon-gamma production, regulation of TNF production)
search/library (Fig. 8E).
generation
Deep learning-based selected
spectra, RTs, and IMs
prediction 4.5 Pitfalls
Protease Trypsin
Missed cleavages default setting used Different protocol steps markedly influence the overall perfor-
Modifications default setting used mance of the workflow and to achieve good, satisfactory results,
Peptide length range default setting used the following pitfalls should be avoided.
Precursor charge range changed to 1–6 One major aspect which is critical for a successful LC-MS
Precursor m/z range default setting used experiment is the sample quality. To avoid any problems arising
Fragment ion m/z range default setting used from low-quality reagents, all (sample) buffers should be pre-
Output pared using either HPLC/LC-MS grade or ultrapure water (pre-
Additional options Command “relaxed-prot-inf” pared by purifying deionized water to attain a conductivity of 18
applied for homology
M cm−1 at 25°C) as well as analytical grade reagents. DDT- and
filtering
Algorithm IAA-containing buffers should always be prepared freshly before
Mass accuracy default setting used use as DDT and IAA are sensitive to oxygen and light, respec-
Unrelated runs default setting used tively. Hence, IAA-containing buffers should be kept in the dark.
MS1 accuracy default setting used Moreover, particularly during FACS analysis and SP3 sample pro-
Use isotopologues default setting used cessing, make sure to work in a clean environment and to always
Scan window default setting used wear appropriate gear (i.e. laboratory coat and nitrile gloves) to
MBR MBR selected minimize sample contamination with keratins derived from dead
Neural network classifier default setting used skin (or hair). For the preparation of LC mobile phases and wash
Protein inference set to “genes species-specific” solutions, exclusively use LC-MS grade solvents and acids as low-
Quantification strategy default setting used quality solvents can markedly impair and reduce the performance
Cross-run normalisation default setting used
of LC-MS experiments. Additionally, make sure that LC-MS sol-
Library generation default setting used
Speed and RAM usage default setting used vents are degassed properly prior to use (see also section 4.2.1.3).
Each sample transfer and processing step can lead to pro-
tein and peptide loss, particularly when working with a limited
replicates and were identified by at least two peptides. The amount of input material. The SP3 procedure already markedly
resulting p-values were corrected for multiple testing using the minimizes processing and transfer steps as compared to other
Benjamini-Hochberg method. Only proteins with an adjusted p- proteomic protocols [41–43]. Nevertheless, to further reduce loss
value below 0.01 and a fold change of at least 1.4 as com- during sample preparation, it is crucial to use protein low-binding
pared to the other groups were included in the list of cDC tubes and pipette tips for sample processing and the collection
subset-specific proteins (Fig. 8). The statistical programming lan- of FACS-sorted cells (see also equipment setup – section 4.2.2.,
guage R including the pheatmap [47], imputeLCMD [48] and Table 13). In the present protocol, cellular lysates are generated
pcaMethods [49] packages was applied to conduct further anal- by directly sorting cDC subsets into an SDS-based lysis buffer (5×
yses and to visualize results (see Fig. 8A and 8B). Functional concentrated). Hence, to achieve optimal lysis conditions, i.e. to
annotation analysis was performed using the DAVID knowledge- ensure that the lysis buffer is not diluted below 1–2% (w/v) SDS
base (Database for Annotation, Visualization and Integrated Dis- and lysis will be efficient, it is critical to estimate the total sort vol-
covery, version 6.8) (https://david.ncifcrf.gov/) [50]. Venn dia- ume prior to the experiment. The total sort volume is dependent
gram data were calculated using the Venny web application on the target number of cells, the size of the nozzle as well as the
(http://bioinfogp.cnb.csic.es/tools/venny/index.html). sorting mode and should be calculated taking these parameters
In total, 4,747 proteins were identified across the whole into account (see also example calculation in section 4.3.2, step
dataset revealing distinct cDC subset-specific differences that 1). We highly recommend to perform a pilot experiment using
undermine the power of integrated FACS and LC-MS analysis the chosen FACS settings. Check if calculated (theoretical) vol-
(Fig. 8). Besides the cell surface markers used for FACS-sorting umes match experimental ones. This way you can ensure that the
(Fig. 8D, left panel) a multitude of additional cell type-specific proper amount of SDS-lysis buffer (5×) required for efficient lysis
proteins could be identified by the proteomic analysis, includ- will be applied in the main experiment. Once cells are collected by
ing different transmembrane proteins and cell-surface receptors, FACS, make sure to directly promote cell lysis by boiling samples
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 FACS isolation and mass spectrometry-based proteomic analysis of rare mouse DC populations 31 of 96
Figure 8. LC_MS analysis of FACS-purified cDCs reveals subset-specific protein expression patterns. Label-free quantitative proteome analysis of FACS-
purified splenic cDC1, cDC2A, and cDC2B cells derived from C57BL/6J mice (n = 5). (A) Heatmap of all quantified proteins in the dataset. For cluster
analysis and heatmap visualization, label-free quantification values were log2-transformed and Z-score normalized for each protein. (B) Princi-
pal component analysis of the transformed dataset identified three distinct clusters along with the first and second principal component cor-
responding to the different cDC subsets. (C) Venn Diagram of differentially expressed proteins in cDC1, cDC2A, and cDC2B (Benjamini–Hochberg
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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32 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
for 5 min (section 4.3.2, step 10) to avoid uncontrolled lysis and carboxylate-functionalized bead mix, and repeat the ACN wash
protein degradation when leaving samples at room temperature and recovery steps. Peptides derived from the initial binding step
for a prolonged time interval. as well as from the second purification step can then be pooled
Another critical step is the initial protein capture to the SP3 prior to MS analysis [41].
beads. Here, several points need to be taken into consideration. A multitude of additional helpful tips and tricks as well as
One is the overall sample volume after sorting (including lysis common pitfalls regarding the SP3 sample processing itself are
buffer), which at best should not exceed 100–120 μL for the discussed in depth by Hughes et al. [42], which we would highly
initial protein binding step in SP3. Hence, we suggest to sort with recommend for further reading.
a nozzle and sorting mode that creates small droplets per cell. In
our hands, working volumes between 30–100 μL and 1–10 μg of
total protein achieved best results for SP3 sample processing [41]. 4.6 Top tricks
Based on the bead binding capacity the ideal SP3 bead-to-protein
ratio is 10:1 (w/w) [43] at a final bead concentration of at least To improve the performance of the present protocol, we recom-
0.5 μg/μL [42, 43]. However, when analyzing limited numbers mend to estimate the protein content in the samples as this has
of FACS-sorted cells these conditions are hard to meet. Typically, a major impact on several protocol steps. One example would be
protein amounts do not exceed 3 μg of protein upon lysis of the LC-MS measurement itself. There is a “sweet spot” for each
10,000-30,000 primary immune cells and could even range below LC-MS setup regarding the optimal sample on-column load that
1 μg. Hence, bead quantity should be adapted to the sample will yield the best results in terms of identified peptides and pro-
volume as suggested by Hughes et al. [42] who recommend a teins as well as quantitative reproducibility. Another example is
working concentration of 0.5 μg beads per 1 μL of processing the proteolytic digest. Knowing the protein content of your sam-
volume in an SP3 sample where protein input material is low. For ple can help estimate the trypsin amount required for efficient
example, to 100 μL of a working (sample) volume 2.5 uL of bead proteolytic digestion. Moreover, too high amounts of trypsin, for
stock should be added, resulting in a final bead concentration of example, can result in abundant trypsin autolysis products that
0.5 μg/μL. Besides the amount of beads, we found that the pH is might dominate and impair LC-MS analysis and results.
a critical parameter during the initial protein-bead binding step. Typically, when sample material is not limited, the total pro-
In contrast to the original SP3 protocol where initial protein cap- tein content of cellular lysates can be determined by colorimetric
ture is performed at acid pH [43], cDC lysates are incubated with protein assays, such as the Pierce 660 nm protein assay (Thermo
the SP3 beads at a neutral pH in the present protocol. As acidic Fisher Scientific). However, when the amount of starting material
conditions can impede protein binding to the carboxylate-coated is limited, as is the case for certain cDC subpopulations, perform-
beads and lead to increased variability [41, 51], we recommend ing a protein assay likely consumes significant amounts of sample
to work at neutral pH. In addition to the protein, intact chromatin prior to LC-MS downstream analysis. In order to estimate the pro-
or RNA can also bind to the magnetic carboxylate-coated SP3 tein content of the actual samples of interest, it is recommended
beads and interfere with the SP3 protocol. In case of the present to perform preliminary experiments either with the cell type of
workflow, we recommend DNA and RNA shearing by means of a interest itself or with similar but less rare sample types such as
suitable ultrasonication device to further aid cell disruption and cell lines (see also note, Section 4.3.3, step 9).
protein extraction. Using a Bioruptor device with the settings To determine the optimal on-column load for LC-MS exper-
described in section 4.3.2, step 12, is typically sufficient to iments, it is also possible to estimate the sample yield at the
shear DNA and RNA. Alternatively, RNA and DNA can also be peptide level performing either fluorometric or absorbance-based
degraded enzymatically, e.g. by benzonase treatment of the cDC assays, such as the Pierce Quantitative Colorimetric Peptide Assay
lysates. (Thermo Fisher Scientific) or measurements at 205 nm /280 nm.
To increase sample recovery, i.e. to make sure that no pep- Alternatively, a pre-measurement of samples can be conducted by
tides are lost after the overnight tryptic digest, optionally the SP3 LC-MS and used to calculate optimal injection amounts for the
peptide purification step can be repeated with the supernatant of main analysis. Besides the sample on-column load, there are a
the initial peptide-binding step (see note, Section 4.3.3, step 12). multitude of additional factors that influence the performance of
To do so, do not discard the supernatant during the first step of LC-MS measurements, which are beyond the scope of the present
peptide purification. Instead, collect the supernatant, add 2 μL of protocol. These include, for example, different LC settings (i.e.

correction p < 0.01, log2-transformed fold change compared to other cDC subsets > 0.5) reveals cell type-specific proteins for each cDC subpop-
ulation. Total number of proteins identified in each subset is indicated. (D) Relative LFQ intensities of selected cDC markers. In the left panel
cDC markers used for FACS analysis are displayed. In the right panel selected markers for cDC1 and cDC2 subsets are depicted. (E) Gene Ontol-
ogy (GO) enrichment analysis of proteins that are significantly associated with either cDC1 or cDC2 subsets (Benjamini–Hochberg correction p <
0.01, log2-transformed fold change compared to the other cDC subset >1).). Full names of GO terms marked with asterisk (*): MHC class I pep-
tide loading complex, antigen processing and presentation of exogenous peptide antigen via MHC class II, antigen processing and presentation of
endogenous peptide antigen via MHC class I, protein disulfide oxidoreductase activity, extrinsic component of cytoplasmatic side of plasma mem-
brane, positive regulation of I-kappaB kinase/NK-kappaB signaling, integrin-mediated signaling pathway, superoxide-generating NADPH oxidase
activity.
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Eur. J. Immunol. 2023;53:2249925 Gelatin degradation assay to address podosome formation and function of mouse DC 33 of 96
Table 15. Overview of mouse splenic cDC phenotypes and cell-specific markers
cDC Surface marker used Markers identified by proteome analysis
subpopulation for FACS analysis
Shared cDC markers Celltype-specific markers
(no differential expression) (differentially expressed)
cDC1 CD11c, MHC-II, XCR1 ITGAX (CD11c), CD45, MHCα BTLA (CD317), CADM1 (NECL2), CD8α,
CLEC9a (CD370), IRF8, ITGAE (CD103),
LY75 (DEC205, CD205), TLR3, Tap1, Tap2,
MHCβ, MHCγ, CD36, ICAM1
cDC2A CD11c, MHC-II, Sirpα, ITGAX (CD11c), CD45, MHCα FCER1, IRF4, ITGAM (CD11b), Sirpα
ESAM (CD172a), TLR71, CD4, RelB, RUNX3,
IKZF1 (IKAROS), Zeb21
cDC2B CD11c, MHC-II, Sirpα ITGAX (CD11c), CD45, MHCα CLEC10a (CD301), IRF4, ITGAM (CD11b),
Sirpα (CD172a), TLR71, CD4, RelB,
RUNX3, IKZF1 (IKAROS), Zeb21, Lyz2
1
ns: close to detection limit – hence t-test not significant, but absent in all cDC1 runs
LC-gradient, flow rate, column type, etc) as well as MS instru- whereas they can be clearly distinguished from other actin-based
ment parameters and MS acquisition modes that might also be adhesive structures involved in cellular migration such as lamel-
instrument dependent. As the samples generated by the present lipodia, filipodia, and focal adhesions (see [56] for an excellent
proteomic protocol can be analyzed on any state-of-the-art instru- overview). We follow this classification and therefore refer to this
ment LC-MS platform, we highly recommend checking the respec- structure in DC throughout this guideline as podosomes.
tive literature or vendor recommendations. Moreover, we advise Podosomes present as dot-shaped, micrometer-sized F-actin
to use data-independent acquisition methods for label-free quan- cores surrounded by an adhesive ring of adhesion plaque pro-
titative proteomic analysis [52–55], as the resulting datasets dis- teins, such as vinculin, paxillin, and talin, and anchored to the
play a significantly enhanced reproducibility and fewer missing ECM by integrins [60, 61]. Meanwhile, it is well-established that
values compared to data-dependent acquisition workflows. podosomes are composed of more than 300 different components,
highlighting their molecular complexity [61]. The characteris-
tic bipartite appearance of the actin core and the adhesive ring
4.7 Summary table facilitates their recognition in immunofluorescence microscopy
(Fig. 9). However, in combination with fluorescent gelatin to
Table 15 lists cDC subpopulation-specific markers used for FACS assess its degradation, the detection of podosomes is limited to
analysis as well as cell-type-specific markers identified by LC-MS the identification of the actin cores.
analysis. A hallmark function of podosomes is the degradation of
ECM that sets them apart from other actin-rich structures [56].
To this end, podosomes recruit matrix-lytic proteases, includ-
5 Gelatin degradation assay to address ing matrix metalloproteases (MMP) and ADAMs (a disintegrin
podosome formation and function of and metalloprotease). However, the role of proteolytic enzymes
mouse DC in other purposes of podosomes remains unclear. Beyond the
above-mentioned features of adhesion and ECM degradation,
5.1 Introduction podosomes are nowadays considered to fulfill additional functions
[60]. The capability of cells to sense the rigidity and topography
Podosomes act as adhesive and invasive structures that are par- of the substrate through the podosomes has been demonstrated.
ticularly abundant in cells of the monocytic lineage including Moreover, podosomes frequently form at protruding sites of the
macrophages, dendritic cells, and osteoclasts but are also found cell. It is thus hypothesized that podosomes are involved in the
in other cell types [56]. Podosomes were indeed first described directional migration of cells by stabilizing otherwise transient
in v-src-transformed fibroblasts as clusters of contact sites of cell- protrusions. This may enable cells to transmigrate across layers
to-extracellular matrix (ECM) adhesion [57, 58]. Chen et al. later of other cells, as shown for osteoclasts. Studies in DC have very
found that degradation of the ECM occurs at these contact sites specifically identified podosome-like protrusions as sites of anti-
and coined the term invadopodia [59]. Nowadays, it is widely gen uptake [60].
accepted to refer to these structures in transformed tumor cells The protocol presented here allows quantifying both
as invadopodia while referring to them as podosomes in normal podosome formation and matrix degradation simultaneously
cells. There are only subtle differences between invadopodia and using fluorescent microscopy and thus can be used to study the
podosomes, for example, regarding the durability of the structure, molecular mechanisms of podosome biology and their function
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34 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Figure 9. Podosome staining and detection in DC. Bone marrow-derived DC were seeded on gelatin-coated coverslips and cultivated overnight
before being fixed and stained for vinculin and actin to detect podosomes (arrows). Images at the top panel were taken with 100x magnification.
The lower panel shows enhanced views of selected areas (white boxes) from the top images to highlight structural details of a podosome. Note
that DAPI staining in the lower panel was excluded for better visualization of the podosome. Scale bar upper panel: 20 μm; scale bar lower panel:
5 μm.
in DC. It is based on a protocol that used fluorescently-labeled 2. Clean and prepare the surface of round 12 mm glass cover-
gelatin as a matrix component for studying invadopodia forma- slips by soaking them in a mixture of EtOH/HCL (6:4; v/v)
tion by invasive tumor cells [62]. The workflow was modified and for 2 h with occasional shaking. Let them air dry. Dried cov-
improved to meet the needs for the analysis of podosome forma- erslips can be stored for later use.
tion and function in mouse bone marrow-derived DC in terms of 3. Sterilize the coverslips by immersion in 70% EtOH solution
gelatin coating, cell seeding, and data analysis. It may also serve and allow to air dry.
as a kernel to extend it for studying further functionalities or of 4. Transfer the coverslips into a 24-well plate and add 500 μl of
other DC subsets, including human monocyte-derived DC [63, poly-L-lysine (50 μg/ml in ddH2O) per well. Incubate at RT
64]. for 20 min.
5. Wash coverslips three times with PBS (optionally, a water jet
pump can be used for aspirating).
5.2 Materials
5.2.1 Reagents Table 16. List of chemicals and reagents
Reagent Manufacturer Catalog
All chemicals and reagents are listed in Table 16. number
Ethanol (EtOH) Merck 1009862500
Hydrochloride acid (HCL) Merck 1090631000
5.2.2 Equipment Poly-L-Lysine, 0.1% in Sigma-Aldrich P8920
ddH20
All equipment is listed in Table 17. Glutaraldehyde 25% Polysciences Inc. 01909
Glycine Merck 1042011000
Phosphate Buffered
Saline (PBS)
5.3 Step-by-step sample preparation
Fetal Calf Serum (FCS) Gibco
RPMI-1640 Gibco 21870076
5.3.1 Coating of coverslips with fluorescently labeled gelatin GM-CSF Peprotech 315-03
Gelatin from pig skin, Invitrogen G13186
1. Prepare fluorescent gelatin stock solution of 1 mg/ml by Oregon Green 488
adding 5 ml of sterile double distilled water (ddH 0) to the conjugate2
commercial vial following the manufacturer’s instructions. DAKO fluorescence Agilent S3023
mounting medium
For coating, a final working concentration of 0.2 mg/ml is
TNFα (optional) Peprotech 315-01A
used. Aliquots of the stock solutions can be stored at –20°C.
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Eur. J. Immunol. 2023;53:2249925 Gelatin degradation assay to address podosome formation and function of mouse DC 35 of 96
Table 17. List of equipment Wash coverslips three times with sterile PBS. From this point
on, the coated coverslips can also be stored in the dark at
Equipment Manufacturer Catalog
4°C in the fridge for up to a week before seeding with cells.
number
Continue to work under sterile conditions.
24-well plate Greiner Bio-One 662160 14. Add 1 ml complete RPMI medium (containing 10% FCS) to
Round 12 mm diameter Fischer Scientific 11708701 each well and equilibrate coverslips at 37°C for 30 min.
glass coverslips
(MenzelTM)
Microscope glass slides Langenbrinck 03-0004
× 5.3.2 Preparation and seeding of DC and performing the(75 25 mm, frosted
end) gelatin degradation assay
Parafilm Bemis PM-996
Forceps Dumont #5 Fine Science 11252-20 1. Harvest bone marrow-derived immature DC from your cell cul-
Tools (F.S.T.) ture and perform a complete RPMI medium change.
Thermomixer set at 2. Count cells using a hemocytometer (or any other cell-counting
60°C device).
Vacuum source (e.g. 3. Resuspend cells at 2×105 cells/ml in complete RPMI medium
water jet pump) for supplemented with GM-CSF (200 U/ml). Optionally add addi-
aspirating (optional) tional factors that you wish to investigate for their influence on
gelatin degradation and the podosome formation by DC, such
6. Add 500 μl of pre-chilled glutaraldehyde (0.5% in PBS) to as TNFα (100 ng/ml), kinase inhibitors, or toll-like receptor
each well and incubate on ice for 15 min. ligands.
7. During this incubation period, the Oregon Green 488- 4. Aspirate the RPMI medium from pre-incubated coverslips and
conjugated gelatin stock solution is heated to 60°C, diluted transfer 500 μl of the DC solution (1×105 cells) per well.
to the final working dilution of 0.2 mg/ml, and kept at 60°C. 5. Incubate for 8–16 h at 37°C.
8. Dispose of the glutaraldehyde properly and wash coverslips
three times with PBS.
9. Place a sheet of parafilm on the bench and spot 50 μl of fluo- 5.3.3 Processing cells for podosome detection and
rescent gelatin working solution on top. Leave enough space fluorescence microscopy
(≥ 1 cm) between the drops. The drops will not disperse and
flow into each other (Fig. 10). All following steps can be again performed under non-sterile con-
10. Coat coverslips by placing them upside down on the fluores- ditions.
cent gelatin solution and leave them protected from light for
30 min at RT (Fig. 10). 1. At the endpoint, wash coverslips once with PBS and quickly fix
11. Transfer the coverslips (with the top side up) back to a 24- cells by adding 500 μl of paraformaldehyde (4% in PBS) per
well plate after the incubation period and let it dry for 10 min well and incubate for 15 min at RT in the dark.
in the dark at RT. 2. Remove the paraformaldehyde and dispose properly and wash
12. Add 500 μl of glycine (0.3 M in PBS; pH7,2) and incubate coverslips three times with PBS.
for 20 min at RT in the dark and wash coverslips three times 3. Add 500 μl of blocking solution (3% BSA in PBS containing
with PBS afterward. 0.1% TritonX-100) and incubate for 30 min at RT in the dark.
13. Transfer the plate with coverslips under the cell culture flow 4. Remove the blocking solution and stain F-actin by adding 180
hood and transfer the coverslips into a sterile 24-well plate. μl Alexa Fluor 594-coupled Phalloidin diluted 1:2000 in 0.3%
Figure 10. Steps for coating glass coverslips with fluorescent gelatin. 1.-2.) Spot 50 μl of pre-warmed fluorescent gelatin working solution on top
of a sheet of parafilm. 3.-6.) Place poly-L-lysin precoated coverslips upside down on the fluorescent gelatin solution. Leave them on for 30 min in
the dark at RT.
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36 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Figure 11. Image analysis using Fiji and quantification of the degraded area per cell. Each channel from the same FOV is used to extract specific
information. The blue channel (A) is used to quantify the number of cells per field by counting the nuclei. The green channel (B) is used to quantify
the area of the degraded gelatin batches. The values obtained from the blue and green channels are used to calculate the degraded area per cell.
The red channel (C) is used to quantify the number of cells that are forming podosome clusters (arrowheads). Scale bar: 50 μm.
BSA/0.1% Triton X-100 in PBS solution per well and incubate 5.3.4 Imaging of podosome formation and activity
for 1 h at RT in the dark.
5. Aspirate the staining solution and wash the coverslips three 1. Images of the slides are taken with a fluorescence micro-
times with PBS. scope, typically using a 40x objective and equipped with filter
6. Add 500 μl of DAPI (1:5000 in PBS) and incubate for 5 min at sets for detection of the green (Oregon Green 488), the red
RT in the dark. (Alexa Fluor 594), and the blue (DAPI) fluorescence. Gelatin
7. Wash coverslips three times with PBS. degradation causes loss of the green fluorescence. Accord-
8. Place a droplet of mounting medium on a glass slide and ingly, black areas in the otherwise homogeneous green back-
mount a coverslip by placing it upside down onto the droplet. ground are indicative of gelatin degradation. The degraded
Let it dry. areas do not have sharp edges and angles, but the bound-
9. Slides can be stored in the dark at 4°C until imaging and for aries appear rather blurred and sometimes speckled (Fig. 11
up to several weeks. and 12).
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Eur. J. Immunol. 2023;53:2249925 Gelatin degradation assay to address podosome formation and function of mouse DC 37 of 96
all DC that might be capable of forming them. Accordingly,
not all DC found associated with degraded areas will present
podosomes.
5.4 Data analysis
All image analyses were performed using the image processing
package Fiji (www.imageJ.net).
1. To quantify podosome formation in DC, open an image with
actin staining and the corresponding image with DAPI stain-
ing. Count the number of cells forming podosomes on each
image of the actin staining and normalize it to the total num-
ber of cells by determining the number of nuclei in the DAPI
staining. Note that some cells show more than one podosome
Figure 12. Squeezing and pulling of fluorescently-labeled gelatin by
DC on a non-precoated coverslip. Images taken in the blue (DAPI), green cluster, while others show none (Fig. 9 and 11). Collected data
(Oregon Green 488-labeled gelatin), and red (Alexa Fluor 594-labeled from all images can be plotted as “percentage of podosome
phalloidin) channels are overlaid as in Fig. 11. Arrows point to squeezed forming cells”.
gelatin deposits. Asterisks indicate degraded areas characterized by
speckled boundaries. However, whether black areas are derived from 2. The gelatin degradation is quantified by analyzing the propor-
gelatin degradation or squeezing is not clearly distinguishable. Scale tion of the entire areas that correspond to degradation on a
bar: 50 μm. given image using the “area fraction” measurement option in
Fiji. Like before, the “area fraction” value will then be nor-
2. Podosomes are visualized in the red channel and identified as malized to the number of cells in each image as measured
ensembles of F-actin dots on the ventral side of the cells in from the DAPI channel in the respective field (Fig. 11). The
contact with the gelatin. degraded area fraction can then be displayed as “percentage of
3. Quantification of podosome formation and activity is per- normalized degraded area per cell”. However, we recommend
formed with the same set of images. To achieve representa- converting the pixel size of the images into metric size (i.e.
tive and unbiased image data we recommend the following μm), which allows plotting the data as, for example, degraded
procedure. Divide the coverslips into imaginary top, middle, area/cell (μm2). The conversion from pixels to the metric unit
and bottom sections and take at least 5 images per section depends on the optical settings of the camera and must there-
in all three fluorescence channels. Survey the sections using fore be determined individually for each device. For the con-
the blue (DAPI) channel and select areas where typically 4–6 version in Fiji go to: Analyze> Set Scale. On the new window
individual nuclei can be identified in the field of view (FOV). enter the respective “Distance in pixels” and the corresponding
Next, acquire images from the same area in the red and green “Unit of length” and confirm with “OK”.
channels. Then, move to another area using again the DAPI For obtaining the “area fraction” value, select Analyze>Set
channel. This allows the most unbiased examination, as FOVs Measurement> and select “Area fraction” from the new
are then not selected primarily for strongly degraded areas or window. Open greyscale images of the green fluorescence
podosome-rich cells. Finally, each sample is represented by a showing the degraded areas as black spots within the gray
set of at least 15 FOV, which in this case represent between at background of the non-degraded gelatin (Fig. 11). Select
least 60 and up to 90 cells. Image>Adjust> Threshold. With opening the “Threshold”
We do not recommend analyzing areas with more than 8 window the image will turn into a black and white image
cells per FOV, since it is very likely that cells at such a high without any grayscale (Fig. 11). Degraded areas may appear
number clustered together, and thus degraded areas cannot as black spots over a white background or as white spots
be attributed to distinct cells. If more cells in such a cluster over a black background. Make sure that the option “Dark
are capable of degrading gelatin this would result in a lower background” is not selected and press “Apply”. If the software
“degraded area fraction/cell” in the calculation (see 5.4 displays black areas that are outside the actual degradation,
Data analysis below) due to the overlap of the degraded for example, due to inhomogeneities of the gelatin coating,
areas. the threshold has to be adjusted manually using the horizontal
IMPORTANT NOTE: A characteristic of podosomes in primary scroll bars in the “Threshold” window. When the dark areas
cells is their fast turnover rate of only a few minutes (2-12 are representative of the real degradation observed in the
min) for assembly and disassembly of the structure. This is original image press “Set”. Record the “area fraction” by select-
in strong contrast to invadopodia of tumor cells, which can ing Analyze> Measure. The data are reported in a “Result”
persist for hours [60]. Consequently, at the time of fixing window and can be saved and transferred to a spreadsheet
the cells on the coverslips, podosomes are not detectable on program.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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38 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
3. Treatment conditions of cells could potentially influence the 6 The under-agarose migration assay: a
adhesion of DC to the gelatin coating. Reduced adhesion may simple method to visualize and quantify
thus result in reduced podosome formation and lower degra- mouse DC migration in confined 3D-like
dation activity. The spread of the cell body on the gelatin coat- microenvironments
ing can be used as a readout of the net adhesive properties of
the cells. The area that is covered by the cell spreading can 6.1 Introduction
be assessed using the same procedure as for determining the
“area fraction” of degraded gelatin (see step 2) but by using On their migration routes dendritic cells (DC) encounter com-
the red channel images of the actin staining. Again, present- plex 3D-microenvironments like the interstitium [65]. The cellu-
ing the data in square micrometers is recommended. lar principles, molecular mechanisms, and mechanobiology [66–
4. Apply the appropriate statistical analysis to determine signifi- 68] of DC migration in 3D-microenvironments can be discovered
cant differences among various conditions. in vitro, such as in collagen matrices [69], microchannels [70, 71],
and forests of micro-pillars [72].
In this section, we describe the ‘under-agarose’ assay [73] as
5.5 Pitfalls an additional, highly simple and versatile [74] method to visu-
alize and quantify DC migration in confined 3D-like microen-
One should be careful not to invert the top side after coating. vironments. The assay (i) can be easily established without
We also recommend the use of very fine forceps, such as micro-engineering equipment, (ii) can be combined with live-cell,
Dumont #5 (or equivalent) to avoid breaking the glass coverslips immunofluorescence, and advanced imaging, (iii) is compatible
when removing them from the 24-well plates. with patterned substrates, and (iv) is suited to investigate sub-
In addition to degrading the gelatin some cells may also pull classes of immune cells like DC subtypes.
the gelatin together (Fig. 12). Since the effect of such squeez- In the ‘under-agarose’ assay, DC migrate between a layer of
ing and pulling cannot be distinguished from the effect of degra- agarose and the substrate (Fig. 13A, B). Thus, DC are highly
dation, in such a case the quantification of gelatin degradation confined and interact with their local microenvironment in three
would be falsified. It was assumed that too strong adhesion of dimensions (in XYZ). Consequently, the locomotion mode is
the cells is the reason for this, and accordingly, means for reduc- closely related to migration in 3D [75]: whereas DC on two-
ing the adhesion of cells to gelatin have been suggested, including dimensional (2D) substrates employ a fundamentally different
reducing serum concentration in the medium or increasing gelatin migration mode that is adhesive and integrin-dependent [76], DC
concentration to 1 mg/ml [62]. IMPORTANT: After we had estab- in the ‘under-agarose’ assay can migrate integrin-independently
lished precoating of coverslips with poly-L-lysine we also noticed without adhesions [77, 78], comparable to low-adhesive in vivo
that the squeezing and pulling effect was minimized and thus we migration in the interstitium [76, 79, 80].
highly recommend this step in our protocol (see also 5.6). While mimicking cellular confinement in 3D, DC in the ‘under-
The ability to form podosomes and their kinetics of assembly agarose’ assay migrate directly on the surface close to the objec-
and disassembly vary depending on the state of differentiation tive. This characteristic enables high-resolution imaging of sub-
and maturation of DC [60, 63, 64]. Maturation by TNFα or toll- cellular processes like cytoskeletal dynamics, for instance by TIRF
like receptor signaling, i.e. using LPS will lead to different results. (total internal reflection fluorescence) [77, 78, 81] microscopy
Maturation with LPS showed a different kinetic of podosome for- (Fig. 13C-E), spinning-disc microscopy [82], and FRET (fluores-
mation in DC dependent on the tissue source, i.e. BM-derived cence resonance energy transfer) [83]. Moreover, the assay is
versus spleen-derived DC [63]. Moreover, LPS stimulation results compatible with immunofluorescent staining [82, 84] and thus
in a complete loss of podosome formation within hours [64]. It STED (stimulated emission depletion) microscopy, as well as
is therefore essential to ensure that endotoxin-free reagents are CLEM (correlated light and scanning electron microscopy) [85]
used. and light-based uncaging of caged chemoattractants [86].
DC in the ‘under-agarose assay’ migrate not only closely but
also perpendicularly to the imaging plane. Thus, multiple cells can
5.6 Top tricks be imaged in low magnification to quantitatively analyze migra-
tion parameters like velocity and persistence [87]. Performing
We recommend using 12 mm round glass coverslips together with the experiment in multi-well dishes with glass-bottoms allows the
a 24-well plate in combination with a water jet pump for all the comparison of different conditions in the same experiment (e.g.,
washing steps. This allows convenient coating and use of up to 24 wild-type versus knockout).
gelatin-coated coverslips in one experiment. Notably, the assay does not require any specialized micro-
Precoating with poly-L-lysine proved to be advantageous in engineering equipment (all reagents and equipment are listed in
several respects. We found that a more homogeneous coating Tables 18 and table 19, respectively) and thus can be established
could be achieved with the fluorescently labeled gelatin. This in every laboratory (Fig. 14A). We here describe the basic proto-
could also be one reason for preventing the squeezing and pulling col of the ‘under-agarose’ assay. However, the assay is highly ver-
of the gelatin coating. satile and thus can be adapted to the respective research question
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 The under-agarose migration assay: a simple method to visualize and quantify mouse DC 39 of 96
Figure 13. The under-agarose assay: imagingmouse dendritic cell (DC) motility in confined 3D-likemicroenvironments. (A, B) Scheme of the setup
of the under-agarose assay.DC (or other cell types) are injected (see also Fig. 14) underneath a layer of agarose (light blue). Thereby,DCmigrate highly
confined and with a flat cell shape between the agarose layer (on top) and the glass surface (below). By stamping a hole as a chemokine reservoir
(see also Fig. 14), the assay can optionally be employed to study DC chemotaxis. (C) Representative DC (Hoxb8-derivedmouse DC encoding the actin
marker Lifeact-GFP) migrating under-agarose and imaged by brightfield (BF), epifluorescence (Lifeact-GFP in green; the nuclear stain Hoechst in
cyan), and TIRF microscopy (Lifeact-GFP in gray). The scale bar equals 10 μm. (D) Zoom into the exemplary regions 1–3 highlighted (orange) in c).
(E) Representative Hoxb8-derived mouse DC (left panel) imaged over the time course of 12 min (middle panel), showing DC shape and locomotion
(right panel). The scale bar equals 10 μm.
(Fig. 14B). Examples include (i) integration of chemokine sources 6.2 Materials
to study chemotaxis or chemokine competition [88], (ii) modula-
tion of the mechanical load by the microenvironment [85], (iii) 6.2.1 Reagents
combination with ligand-patterned substrates [80, 89], and (iv)
combination with substrate topographies like nano-ridges [85, 90, Necessary reagents are listed in Table 18
91]. Moreover, the assay is compatible with other leukocytes like
neutrophils [92], neutrophil-like HL-60 [93] and PLB-985 cells 6.2.2 Equipment
[94], T cells [90, 95, 96], and non-leukocytes like endothelial cells
[97], cancer cells [98, 99], zebrafish progenitor cells [100], and Necessary equipment is listed in Table 19
the single-cell amoebae Dictyostelium [101]. As only small cell
numbers are required, the assay can be combined with leukocyte-
pathogen studies [102] and immune cell isolations from patients 6.3 Step-by-step sample preparation
with human immunodeficiency [103].
In summary, the ‘under-agarose’ assay is a simple but highly 6.3.1 Preparation of the ‘under-agarose’ assay
versatile assay to investigate the cellular principles and molecular
mechanisms of DC migration in confined 3D-like microenviron- 1. Preparation of reagents: Prepare 2× HBSS by diluting 100
ments. ml of 10× HBSS in 400 ml of ddH2O. As a precaution, the
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40 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 18. Reagents and solutions Table 19. Necessary equipment
Reagent Manufacturer Catalogue Equipment Manufacturer Catalogue
Number Number
RPMI 1640, no phenol red Gibco 11835063 μ-Slide 8 Well Glass Ibidi 80827
Fetal Calf Serum (FCS) Gibco Bottom
10,000 U/ ml Penicillin- 10 Sigma-Aldrich P0781 35 mm glass bottom Greiner 627860
mg/ ml Streptomycin dish Bio-One
(P/S) Harris Uni-Core puncher Sigma-Aldrich WHAWB100076
2-Mercaptoethanol (50 mM) Gibco 31350-010 2.0 mm (optional)
UltraPureTM Agarose Invitrogen 16500-500 Water bath, adjustable
10× Hank’s Balanced Salt Thermo Fisher 14065056 to 55°C
Solution (HBSS) Scientific Microwave
CCL19 (optional) bio-techne 440-M3
NucBlue (optional) Invitrogen R37605
P/S and 660 μl 2-Mercaptoethanol to 500 ml RPMI-1640.
R20 and 2× HBSS can be stored at 4°C for later use.
solution may be filter-sterilized and stored at 4°C. Prepare 2. Decide for the final agarose concentration (influencing
RPMI-1640 + 20% FCS (R20) by adding 120 ml FCS, 6 ml the stiffness of the agarose gel [85]) and calculate the
Figure 14. Graphical overview & versatility of the protocol to visualize and analyze DCmigration. (A) Graphical step-by-step protocol to investigate
DCmigration in the under-agarose assay (see themain text for experimental details). (B) Graphical overview on the versatility of the under-agarose
migration assay.
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Eur. J. Immunol. 2023;53:2249925 The under-agarose migration assay: a simple method to visualize and quantify mouse DC 41 of 96
ingredients accordingly. We routinely use a final agarose 6.3.2 Injection of DC into the ‘under-agarose’ assay
concentration of 1% when investigating DC: calculate the
ingredients in a 1:2:1 ratio (2× HBSS:R20:water-agarose) 1. (Optional) Combine the under-agarose assay with chemotaxis:
for a final agarose solution with a concentration of 1% Stamp a hole into the agarose layer (e.g. by using a biopsy
agarose (e.g. use 5 ml 2× HBSS, 10 ml R20, and 5 ml puncher with 2 mm diameter) and fill the hole with the respec-
4% agarose for a total volume of 20 ml). Note that the tive chemokine (e.g. 15 μl of 2.5 ng/μl CCL19 diluted in
components of the agarose solution have to be added in a medium; see also Fig. 14).
sequential order (steps 3–5). 2. Harvest DC (e.g. bone-marrow derived LPS-matured DC) from
3. Start to prepare the agarose solution by first mixing 2× HBSS your culture and count the cells.
and R20 in a 50 ml falcon tube and pre-equilibrate in a water 3. Inject 20,000 DC dissolved in 1 μl media between the glass-
bath to 55°C (30 min of pre-equilibration; see also Fig. 14). coverslip and the agarose-layer by using smallest pipette tips.
4. Dissolve the agarose powder in ddH2O in another 50 ml Be careful to push the pipette tip entirely through the agarose
falcon tube (e.g., at an initial concentration of 4% agarose layer until you reach the glass surface: hold the pipette straight
to achieve a final concentration of 1% agarose). Heat up and only then release the 1 μl of cell solution while still push-
by using a microwave until the agarose is fully dissolved: ing mildly against the glass slide. If you are combing the assay
heat shortly, shake/vortex the tube and heat up again in with a chemokine gradient (step 1), inject the cells in 5 mm
the microwave; repeat these steps at least three times until distance to the chemokine hole 1 h after adding the chemokine
the agarose is completely dissolved (important: (i) mildly (this allows the chemokine gradient to be established by diffu-
unscrew the falcon lid and (ii) prevent overheating by reduc- sion).
ing microwave power). 4. (Optional pause point): Before imaging, we recommend to
5. Immediately pour the 55°C pre-equilibrated 2× HBSS/R20 incubate the DC upon injection for 2 h below the agarose layer.
mixture (step 3) to the dissolved agarose (step 4); see also Depending on the research questions, longer incubation peri-
Fig. 14. ods in the range of several hours are also possible. Thus, this
6. Immediately pipette the mixture from step 5 into an imaging- step represents an optional pause point, if necessary. Yet, we
suitable dish with glass bottom (e.g., use 2.5 ml for a 3 cm do not recommend incubation for very long time periods (e.g.,
glass bottom dish or 300 μl/well for an 8-well IBIDI μ-Slide). overnight or for several days), as long-term confinement might
7. (Optional): Usage of coated glass-coverslips: If you use a induce changes in gene expression and alterations in the DC
coated glass surface (e.g. PEG-, Fibronectin-, poly-L-lysine- differentiation/maturation status (see also ‘pitfalls’, point 4).
or ConA-coated), then let the 2× HBSS/R20/water-agarose
mixture (step 5) cool down to 37°C before adding it onto the
coated coverslips to prevent inactivation of the coating. 6.3.3 Live-cell Imaging of DC in the ‘under-agarose’ assay
8. (Optional) Inhibitor experiments: the under-agarose assay
can be combined with pharmacological inhibitors. To prevent 1. Use an inverted microscope with an incubation chamber
inactivation of the inhibitors, let the 2× HBSS/R20/water- heated to 37°C and supplied with humidified 5% CO2. Setup
agarose mixture (step 5) cool down to 37°C until you the microscope and the microscope software according to your
add the inhibitor in the respective concentration into the research question.
HBSS/R20/water-agarose mixture. Do not forget to add 2. (Optional) DIC microscopy: as the agarose-layer inter-
the inhibitor also in the later steps to the cells and to feres with DIC microscopy, adjust the amount of the 2×
the chemokine to ensure a homogeneous concentration of HBSS/R20/water-agarose mixture to only result in a thin layer
the inhibitor. of agarose.
9. Solidify the agarose mixture for 1 hour (h) at room tempera- 3. (Optional) Instead of live-cell imaging, the under-agarose
ture to generate a solid agarose gel. assay can be combined with fixation and thus immunofluores-
10. Equilibrate the entire set-up at 37°C and 5% CO2 in a humi- cence staining (see e.g., [82, 84]). Note that the fixation agent
fied cell culture incubator for 1 h. has to diffuse through the agarose layer; we recommend to
11. (Optional pause point): We recommend to continue with the add 37°C pre-warmed 4% PFA followed by incubation for 1 h
next step (injection of DC) after equilibration of the set-up at 37°C.
for 1 h. Yet, the set-up can also be incubated for longer time
in the incubator (e.g., 2 to 5 h). Thus, this step represents an
optional pause point, if necessary. However, we do not recom- 6.4 Data analysis
mend long incubation (e.g., overnight), as changes in humid-
ity can influence the assay during long incubation periods DC migrating in the ‘under-agarose’ assay can be analyzed by mul-
(e.g., we occasionally observed (i) drying-out of the agarose tiple open-source- and commercial-image analysis software pack-
layer, if the humidity is too low in the cell culture incubator, ages. Below we describe the analysis of basic DC migration param-
and (ii) floating of the agarose layer, if the humidity is too eters using the image processing package Fiji/ImageJ [104, 105]
high in the cell culture incubator). (www.imageJ.net).
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42 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
1. To quantify basic cell migration parameters (e.g., migration 4. We typically employ the ‘under-agarose’ assay for short-term
speed or directionality), cells can be tracked and analyzed investigations (2 to 5 h) and with LPS-matured mouse DC.
using the Chemotaxis and Migration Tool [106] plug in. When employing non-matured DC or DC from tissues, it is crit-
If a live-cell compatible nuclear staining is included (e.g., ical to consider that changes in gene expression patterns and
Hoechst), cells can be tracked automatically using the Track- in the differentiation/maturation status can occur when DC
Mate [107] plug in (select Plugins > Tracking > TrackMate). are extracted from tissues. Thus, we recommend to either (i)
Detailed information on how to use TrackMate can be found only perform short-term investigations in the range of a few
here TrackMate-manual.pdf. If the experiment was performed hours directly upon DC isolation, or (ii) carefully control the
without fluorescent staining, cells can be tracked manually by differentiation/maturation status when performing long-term
selecting Plugins > Tracking >Manual Tracking. The tracking investigations. Similarly, for long-term investigations we rec-
data are reported in a ‘Result’ window and include information ommend to carefully exclude any endotoxin contaminations
on track number, slice number, as well as X and Y coordinates. that could alter the DC maturation status.
Import the respective tracking data table in the Chemotaxis 5. DC are highly confined in the under-agarose assay. Thus, DC
and Migration Tool [106] plug in (select Plugins > Chemotaxis typically drag their nucleus – the largest and stiffest organelle
Tool> Import data) and initialize the data under ‘Settings’ by – rather at the back of the cell, whereas DC in less confining
specifying X/Y units and time intervals according to your imag- microenvironments position their nucleus rather to the front
ing setup. If desired, tracks with a distinct track length can be of the cell body directly behind the lamellipodium.
specifically selected for downstream analysis by adjusting the 6. DC migration is typically highly sensitive to temperature and
parameters for ‘Number of slices’ accordingly. Add the dataset CO2. Thus, we highly recommend using microscopes with
and apply the settings. Once initialized, tracks can be plotted, incubation chambers pre-heated to 37°C for at least 1 h and
for example, on an X/Y diagram or as rose plot. For obtaining supplied with humidified 5% CO2
additional parameters such as directionality, velocity, accumu-
lated or Euclidian distance, select the ‘Statistic feature’ tab.
2. To analyze the cell shape, the ‘Particle Analyzer’ can
be employed: Open an 8-bit greyscale image of a 6.6 Top tricks
fluorescent staining visualizing the entire cell. Select
Image>Adjust>Threshold. Thereby, the image is converted into 1. Be careful when dissolving the agarose powder in water by
a binary image. Make sure that “Dark background” is selected. using a microwave: too frequent/long heating periods could
If necessary, adjust the threshold manually using the scroll result in water desiccation and thus a higher final agarose con-
bars until the binary image is representative of the original centration than planned.
greyscale image and press “Set”. Select Analyze>Analyze Par- 2. We recommend using sufficiently large volumes (at least
ticles. Adjust size parameters according to cell size and confirm 5ml) to dissolve the agarose powder in water. Otherwise,
with “OK”. The data are reported in a “Summary” window and the water might desiccate too quickly and thus would again
include parameters describing cell shape, e.g., area, major and alter the final agarose concentration. Always use tubes/
minor axis, and circularity. If it is not possible to generate a beakers 2–4 times the volume of the agarose solution to be
representative binary image due to a high background signal, prepared.
we recommend performing segmentation with Ilastik [108], 3. We recommend ensuring that agarose gels are equilibrated at
a free open-source software for image classification and seg- 37°C always for the same duration (1 hour) for every replicate,
mentation. since longer incubation may lead to desiccation of agarose gels
and thus would alter the degree of cellular confinement by the
agarose.
6.5 Pitfalls 4. For live-cell imaging, we recommend adding a reservoir of
water around the sample to avoid desiccation of the agarose
1. Desiccation/humidification of the agarose gel will affect the gel (even if humidified CO2 is used). For example, when using
degree of cellular compression. Thus, in each step of the pro- multi-well plates, we recommend adding water into the empty
tocol one should take care that the agarose neither dries out space between the wells.
nor gets too watery (see ‘top tricks’ 1–4). 5. Prepare fresh agarose gels prior to every experiment. On the
2. Inhomogeneous dissolving of the agarose powder would result same day, prepared agarose gels may be kept at room temper-
in agarose clumping, thereby effecting light scattering and ature for a few hours before start of experiment.
thus imaging properties (see ‘top tricks’ 5–7). Similarly, a very 6. Allow agarose powder to hydrate in cool H2O for a few min-
thick layer of the agarose gel would reduce the quality of the utes before heating in microwave. This allows dispersion of
optical properties. agarose powder and easier dissolution.
3. Be careful to not destroy coated surfaces or pharmacological 7. Avoid too vigorous vortexing of agarose to prevent particles
inhibitors by temperatures above 37°C (see 6.3.1, points 7 and from sticking to the wall of the tube, as this may result in
8). undissolved agarose particles in the final product.
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Eur. J. Immunol. 2023;53:2249925 Quantification of DC migration in 3D collagen matrices 43 of 96
Figure 15. Scheme showing the guidance mechanisms induced by CCR7 activation. CCL19 stimulates DC chemotaxis (left panel) by generat-
ing a soluble gradient. CCL21 bounds to the extracellular matrix generating an adhesive gradient, stimulating DC haptotaxis along immobilized
chemokines.
8. If you encounter air bubbles after combining 2× HBSS with motif chemokine 19 (CCL19) and C-C motif chemokine 21
dissolved agarose, then return the mixture to the 55°C water (CCL21), which interact differentially with the extracellular
bath for 2 min to let bubbles rise to the upper surface. matrix (Fig. 15), but in both cases induce directional migration
9. Glass-bottom imaging dishes are commercially available in dif- [88, 118]. CCL19 remains preferentially in soluble form (Fig. 15,
ferent sizes (e.g., 8-well μ-Slides from IBIDI, 3.5 cm dishes left), and therefore it induces chemotaxis [88, 118]. On the other
fromMatTek, or 3 cm glass bottom dishes from Greiner). Alter- hand, CCL21 released from the lymphatic vessels binds to the
natively, custom-made dishes can be used, enabling the use of extracellular matrix [88, 118], and establishes an adhesive gra-
multi-well dishes like 6-wells: drill circular holes (e.g., made dient (Fig. 15, right). The latter is named haptotaxis, which refers
by the institute’s workshop) into the bottom of the plastic dish to the sensing of surface-bound chemical cues [88, 118].
and cover the hole with a cover glass suitable for microscopy In the recent years the number of techniques to study spon-
(glued to the dish by e.g., paraffin). taneous DC migration has increased [68, 70, 71, 88, 111, 118-
120]. Oppositely, the study of directional DC migration has lim-
ited possibilities and still one the most used techniques to study
chemokine-induced migration is the transwell assay. However,
7 Quantification of DC migration in 3D despite its advantages this technique does not allow the monitor-
collagen matrices ing of cell behavior dynamics, and it only evaluates the transmi-
gration capacity of the cells. Then, the use of microscopy for live
7.1 Introduction cell imaging emerges as a powerful tool to evaluate DC migration.
In particular, in order to mimic the topology of the complex 3D
Dendritic cells (DC) patrol tissues in search for danger signals microenvironments in which DC navigate in the tissue [88, 118],
in health and disease [65, 109]. These signals include damage- the use of 3D matrices has emerged as great alternative [121].
associated molecular patterns (DAMPs), such as extracellular In this section we describe, a simple method to visualize DC
adenosine triphosphate (ATP) [110, 111]. In addition, DC are acti- migration in 3D microenvironments. In addition, we take advan-
vated by pathogen-associated molecular patterns (PAMPs), such tage of the features of CCL19 and CCL21, to study both chemo-
as bacterial lipopolysaccharide (LPS) [87, 110-113]. Interest- taxis and haptotaxis. The current section shows the protocol to
ingly, ATP and LPS activate different calcium signaling pathways, study mouse DC, but the same protocols can be used to study
but both require actin cytoskeleton reorganization to increase human DC.
DC migration [68, 114], revealing the functional convergence
of DAMPs and PAMPs [110, 115]. Moreover, both danger sig-
nals trigger the expression of chemokine receptors (i.e. C-C 7.2 Materials
chemokine receptor type 7, or CCR7), which allow DC traffick-
ing toward the lymphatic vessels [116]. The overexpression of 7.2.1 Reagents
CCR7 after encountering DAMPs and PAMPs highlights the rel-
evance of chemokine sensing and DC migration to initiate the Necessary reagents are listed in Table 20.
adaptive immune response [109]. Consequently, the distortion of
both DC migration and CCR7 signaling is associated to diseases
[116, 117]. 7.2.2 Equipment
CCR7 binds two chemokines of the same family, but that
have different properties [116]. These chemokines are C-C Materials for imaging of collagen assay are listed in Table 21.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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44 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 20. List of reagents specific for DC culture and collagen assay it is recommended to use 500 μM to observe an increase in the
speed of migration as previously described [111]. In the case
Reagent Manufacturer Catalog
of LPS it is recommended to use 100 mg/ml, although lower
Number
concentrations (>10 ng/ml) also trigger DC maturation after
BMDC overnight treatment.
Iscove Modified Dulbecco ThermoFisher 31980048
Media (IMDM) Glutamax
2-Mercaptoethanol (50 ThermoFisher 31350010 7.3.2 Preparation of collagen gels
mM)
Fetal Bovine Serum (FBS) bioWest(VWR) S1810-500
Penicillin/Streptomycin ThermoFisher 15070063 In this article, we present a protocol to prepare two different types
(P/S) of collagen matrices, which have different features as described in
GM-CSF R&D Systems 451-ML/CF the sections below.
IL-4 R&D Systems 404-ML/CF
DPBS ThermoFisher 14190169
EDTA ThermoFisher 2187281 7.3.2.1 Bovine collagen type I.
Lipopolysaccharides from Sigma L6S11
Salmonella enterica 1. The preparation of the mixture with bovine collagen is per-
serotype typhimurium formed on ice, in order to prevent early polymerization. Thus,
Collagen I, Rat Tail Corning® 354249 add 13.5 μl of the media to a 500 μl tube. For a detailed
Collagen I, bovine Neutragen 5010 description of reagents see Table 21.
Advanced 2. Carefully add 102 μl of bovine collagen type I to the media
Biomaterials without mixing (stock of 3 mg/ml).
NaOH (Sodium hydroxide Sigma (Merck) 1091371003 3. Add 60 μl of cells (cell suspension contains 2.7×106 cells/ml)
solution)
and homogenize carefully.
NaHCO3 (Sodium Sigma (Merck) S8761-500ML
bicarbonate solution) 4. Finally, add 6.5 μl NaHCO3 (stock solution 7.5 %) and mix
Silicon paste Avantor 291-1220 gently to avoid the formation of air bubbles in the solution.
rmCCL19/MIP-3β R&D Systems 440-M3/CF
rmCCL21/6Ckine R&D Systems 457-6C/CF
DPBS (10×) ThermoFisher 14200075 7.3.2.2 Rat tail collagen type I.
5. First, add 12.5 μl of PBS (10×) to a 500 μl tube.
Table 21. Materials specific for imaging of collagen assay 6. Place 29.4 μl media on top. Then carefully add 34.2 μl of rat
Equipment Manufacturer Catalogue Number collagen (stock of 3 mg/ml).
7. Add 45 μl of cells (cell suspension contains 2.7×106 cells/ml)
Sterile glass wpi-europe FD35-100 8. Finally add 3.9 μl NaOH (1 M) to start the polymerization.
bottom dishes, Mix the sample carefully until it is homogeneous.
35.5×10mm
Glass coverslips Mercateo CB00120RA120MNZ0
7.3.2.3 Preparation of experiment setup.
7.3 Step-by-step sample preparation 9. Prepare a bottom glass Petri dish (Fluorodish) by placing 3
drops of silicone forming an equilateral triangle (Fig. 16A).
7.3.1 Generation of bone marrow derived mouse dendritic For a detailed description of the materials to prepare this and
cells following steps, see Table 21.
10. Add 40 μl of the collagen/cell mixture (from either 3.2.1 or
1. DC precursors from mice bone marrow were differentiated 3.2.2) to the center of the three silicone drops.
into bone marrow-derived DC (BMDC) with cytokines (50 11. Using tweezers place a 12 mm circular glass coverslip on top
ng/ml GM-CSF and 50 ng/ml IL-4). The cultures were main- of the collagen/cell mixture so that the border sticks to the 3
tained in IMDM media supplemented with FBS (10%), Peni- silicone droplets (Fig. 16A). Push the coverslip gently so the
cillin/Streptomycin (100 U/ml), and 2-mercaptoethanol (50 silicone is stuck in the coverslip.
μM) for 9–10 days, as previously described [111]. For a 12. Prevent the collagen/cell mix from leaking outside the bor-
detailed description of reagents see Table 20. der of the coverslip (Fig. 16A). In addition, the collagen/cell
2. To activate DC and induce their maturation and expression of mix should not leave empty spaces below the surface of the
CCR7, it is possible to use a short pulse (30 min) of danger sig- coverslip to prevent the drying of the sample (Fig. 16A).
nals and analyze the cellular behavior after 16 h (overnight). 13. Incubate the collagen/cell mix for 20 min at 37°C to allow
For ATP concentration >100 μM ATP trigger full maturation, collagen polymerization.
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Eur. J. Immunol. 2023;53:2249925 Quantification of DC migration in 3D collagen matrices 45 of 96
Figure 16. The collagen assay, imaging DC migration in 3D. (A) Scheme of the setup and procedure to prepare a collagen assay. A cover glass
is placed on top of the silicone drops and collagen mix. The edge of the cover glass should adhere to the silicone and the collagen/cell mixture
should fill the entire cover glass. Avoid to add an excessive amount of collagen/cell mixture or to push the coverslip too strongly. On the other
hand, prevent leaving empty spaces below the surface of the coverslip by adding a lower amount or by failing to push the coverslip. (B)Microscopic
images (10× magnification, phase contrast) of bovine and (C) a rat collagen gel. Note that bovine collagen forms large bundle/fibers that might
steer the DC migration during directional migration. Unlike bovine, rat collagen shows a much more homogeneous mixture. The inset on the right
shows a microscopic image (40x magnification, phase contrast) the rat collagen fibers randomly organized, mimicking the extracellular matrix of
a tissue. Calibration bars of 100 and 20 μm are depicted.
14. Upon collagen polymerization, carefully add 2 ml of media. 7.4.1 Analysis of DC migration in collagen (3D
Add chemokines accordingly. Use >100 ng/ml CCL19 to ana- environments)
lyze the chemotaxis, and >150 ng/ml CCL21 for the hapto-
taxis of DC. The suggestion is to use equimolar concentra- 7.4.1.1 image processing.
tions of both chemokines to have at least 10 nM.
15. To analyze dynamic cell behavior perform live cell imaging.
To monitor cell migration in a large number of cells low mag- 1. Rotate the movie so the cells migrate horizontally from left
nification (10×) acquisition is enough (Fig. 16B and C). Use to right (according to the gradient). To found the right angle
a system equipped with controlled atmosphere (5% CO2 and of rotation, highlight the migration of cells using Z-Projection
37°C). It is recommended to use phase contrast to better visu- (Stk - Z Project - Max Intensity). This will show a time pro-
alize the migration of cells, but bright field is also compatible. jection of the trajectories and will help to define the rotation
A frame rate of 2 min is enough to analyze overall DC migra- angle.
tion. Faster imaging is also possible but it increases the file 2. Crop the region of interest to x μm from the edge of the cover-
size and might limit the amount of positions. slip. This zone should be defined according to gradient shape
16. For detailed analysis of DC shape during migration, 40x mag- and stability, but it is recommended to use a 250 μm region
nification and higher time frame acquisition (every 10 sec) close to the chemokine source.
are recommended (Fig. 16C). 3. To optimize the analysis of migration, it is recommended to
17. Pause point. Once the collagen is polymerized and the cells first apply several filters as follows.
are flooded with media, the samples could be kept for a few 4. First create an average time projection of the movie (Stk - Z
hours in the incubator until imaging. Projection - Average Intensity) and subtract it from the previ-
ously rotated movie (Process - Image Calculator). This step
will remove the sessile cells from the analysis leaving only
7.4 Data analysis motile cells as white objects over a dark background.
5. Duplicate the processed movie and apply a Mean Filter of 20
The analysis of DC migration is performed using Fiji (Image J) (Process - Filters - Mean Filter). The radius should be adjusted
software. depending on the cells used (DC: 20 μm). Subtract the gener-
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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46 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
ated image from the movie (as described in step 4), to remove 9. In the next dialogue box, the results of tracking will be dis-
light reflections from the cells. played. Color-coded tracks will appear, showing the progres-
6. Finally, apply a Gaussian Blur filter of 5 μm (Process - Filters - sion of the spots across the video. Here, tracks could be
Gaussian Blur). In order to visualize the cells in the processed filtered according to the experimental parameters defined
images, adjust the brightness/contrast needs to be adjusted based of the sample’s behavior. For DC it is suggested to
accordingly. The Gaussian filter causes a round white shape choose tracks longer than 10–20 min depending on the acqui-
of the cells that can be easily tracked by the image software sition frame rate to have enough information about the
analysis. migration of each cell. From the drop menu choose “Track
duration” and adjust to 1200 or according to your sample.
10. In the last step the data is obtained. Click the button “Tracks”
and export the sheets “Spots” and “Tracks” as CSV. These
7.4.1.2 cell tracking.
tables contain several parameters including: Track displace-
ment, Track mean speed that could be plot in other soft-
1. For tracking migrating cells use TrackMate v7 plugin from Fiji ware (Fig. 17A-C). A very useful tool is the color-coding of
(ImageJ) [107, 122]. Open the video created in 4.1.1 and the tracks and their appearance along the movie of their full
start the tracking plugin (Plugins - Tracking - TrackMate). display (Fig. 17A). Once a visualization parameter is chosen
2. In the first options panel adjust the spatiotemporal calibra- the merged video containing the tracks and the cells can be
tion of your data. This information is normally provided exported in the next windows.
when reconstituting the movies using .nd files or it is given by 11. From the “Display Options” panel move “Next” two times
the software during image acquisition. Click “next” to move until the panel “Select an action”. Choose “Capture overlay”
to the following window. from the drop menu and export the movie by choosing the
3. In the next section, select a detection algorithm, offering a first and last frame (Fig. 17A). It is also possible to export
choice of 3: The Log detector is optimal for examining spot only the track by selecting “Hide image”.
sizes between approximately 5 and 20 pixels in diameter (use 12. For further information visit https://imagej.net/plugins/
this algorithm for DC). Use the DoG detector for small spot trackmate/ and read the article by Ershov et al [122].
sizes less than approximately 5 pixels and the downsample
LoG detector for spots larger than around 20 pixels in diam-
eter. 7.4.1.3 Visualization of DC migration.
4. Next, add an estimated blob diameter of 20 for DC. If
required, enter a numerical value in the “Qualitative thresh- 1. Use the chemotaxis and migration tool 2.0 from Ibidi to visu-
old”, although is preferable to avoid filtering object detec- alize the tracking data.
tion at this stage. Once optimal segmentation parameters are 2. Open the table “Spots” obtained in 4.1.2 (step 10). Make a
defined, the segmentation is started with the “next” button. new table containing the data track ID, slice, x and y coor-
5. In the next window it is possible to adjust the performance, dinates, or the data will not be uploaded. The first column
memory and hard disk usage. This step is only necessary for should not start with the track ID. Remove dots and commas.
a very large number of spots (more than 1 million). For a Only the first row might contain the name of the parameter.
smaller number of spots, just hit the “next” button. Save the table as a .txt file.
6. In the next window named “Set filters on spots” the shape 3. Go to Migration tool 2.0 and “import data”, choose the .txt file
of violet circles with the previously defined diameter should from step 1.
appear. Irrelevant spots could be now removed. To do so, 4. Initialize the data under settings by specifying X/Y units and
press the “+” button to add a feature filter. Select “Qual- time intervals according to your imaging setup.
ity” filter. A small box should appear at the top containing 5. Once initialized, tracks can be plotted. Use the diagram plot
a histogram. To move the threshold, click and drag in the his- “Rose diagram” (apply settings - diagrams - Plot “Rose dia-
togram window. Choose 0.1 as filter value but adjust accord- gram”) (Fig. 17D). This plot can be saved by right clicking
ingly to your samples by verifying the right detection of spots on the diagram. Comparison between random and chemokine-
in the image. induced migration is useful to evaluate changes in DC direc-
7. To create tracks with the selected spots, a compatible particle tional migration.
linking algorithm can be selected in the next window. Use 6. More information is contained in the Instructions of Chemo-
the tracker “the nearest neighbor”. Here, each spot in one taxis and Migration Tool 2.0 (ibidi.com).
image is linked to another in the next image. You can set
a maximum linking distance, which is indicated in the next
window. 7.5 Pitfalls
8. Add a maximum linking distance of 20 pixel, defined for
the optimal diameter of an average DC spot. The log panel 1. After activation of the cells with LPS, the supernatant should
appears, in which the tracking process is shown. carefully collected ensuring to keep the semi-adherent cells,
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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Eur. J. Immunol. 2023;53:2249925 Quantification of DC migration in 3D collagen matrices 47 of 96
Figure 17. Analysis of DC migration in 3D. (A) Microscopy image of a field of view showing the migration tracks of DC undergoing chemotaxis in
CCL19 gradient for 5 h. A region close to the chemokine source is highlighted with the dotted rectangle. A similar field of view obtained during DC
haptotaxis towards CCL21. Note that on the left side of the field of view, DC migration remains random. (B) Graph showing the speed of migration
induced by CCL19 and CCL21 in bovine or rat collagen (3 mg/ml). (C) Graph showing the cell displacement in the same conditions shown in (B). N =
3 independent experiments, n= 500 cells. (D) Rose plots depict the randommigration of DC in absence of chemokine, and the directional migration
towards the chemokine source (on the right side).
and the flushing should be done gently to avoid recovering 5. If the chemokine will be added during the experiment acutely,
the adherent cells that do not migrate. prepare a stock of at least 100 μl in order to ensure proper
2. Prepare the mixture with bovine collagen keeping everything diffusion and a quick effect during the duration of the experi-
on ice to prevent premature polymerization. Likewise, avoid ment.
desiccation of the collagen mixture, as this could decrease cell 6. Use very fine forceps to avoid breaking or damaging the glass
viability and/or change the diffusion of chemokines. cover, as this will result in the presence of debris during the
3. Avoid the formation of bubbles during the preparation of the microscopy.
collagen mix, as these interfere with the migration of cells and 7. The Chemotaxis and Migration Tool 2.0 provides very useful
distort the tracking analysis. analysis for directionality, which are otherwise not available
4. Prevent freezing and thawing cycles of chemokines, in open access software. However, this software is not very
because this could result in reduced effects in directional user friendly, not intuitive, and the annotation of instructions
migration. is deficient.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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48 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
8 Analyzing mouse skin DC migration by cytometric analysis of the distinct cutaneous DC subtypes using a
whole skin explant culture and FITC conserved core marker antibody panel.
painting assays
Table 22. General reagents for crawl-out and FITC assay
8.1 Introduction Reagent Manufacturer Catalogue
number
Dendritic cells (DC) are central players of the immune system
RPMI-1640 + L-Glutamin Gibco 21870076
as they initiate and modulate immune responses to invading Fetal Bovine Serum (FCS) Sigma F7524
pathogens. Therefore, DC are strategically positioned at the bor- Phosphate Buffered Saline Sigma D8537
ders to the environment, like the skin, where they form a dense (PBS) without calcium
network to maintain cutaneous immune homeostasis [123, 124]. and magnesium
Upon pathogen/ antigen (Ag) recognition, DC mature from highly Ethylendiamintetraacetat Sigma E5134-500G
phagocytic cells into professional Ag-presenting cells along with (EDTA) (0.5M)
their migration to tissue-draining LN, where they prime naïve T FC Block (Anti-CD16/32, BioxCell BE0307
cells and initiate adaptive immune responses. Next to their crucial Anti-FcvRIIB/777)
role in protective immunity, DC are also essential in maintaining Cytofix/Cxtoperm BD 51-2090 KZ
immune tolerance towards self- and innocuous environmental Ag Perm/Wash buffer BD 51-2091 KZ
Trypan blue Gibco 15250-061
[125]. To fulfill these opposing tasks, the skin accommodates phe-
notypically and functionally specialized DC subtypes that can be
distinguished by their differential expression of integrins, such as Table 23. General solutions for the crawl-out and FITC assays
CD11b and CD103, as well as the C-type lectin receptor Langerin
(CD207) [124]. Solution Ingredients End concentration
Langerin+ Langerhans cells (LC) are epidermis-resident Ag- PBS/ EDTA solution PBS
presenting cells. Although LC are Flt3L-independent and, like FACS buffer EDTA 2 mM EDTA
many tissue-resident macrophages, of embryonic origin and self- PBS
maintain in situ, functionally they represent typical DC in that EDTA 2 mM EDTA
they migrate to draining lymph nodes (LN) and activate naïve FCS 3 %
Ag-specific T cells [126]. LC express cell-adhesion molecules such
as EpCam (CD326) and E-cadherin, which allow them to form Table 24. Specific reagents for the crawl out assay
close cell contact with the surrounding keratinocytes [127]. More-
over, LC are the only APC population in the epidermis where they Reagent Manufacturer Catalogue
create the first line of defense against invading pathogens [128], number
but can also negatively regulate protective immunity during infec- Penicillin/Streptomycin Gibco 15140-122
tion [129]. In contrast to the epidermis, the dermis harbors dif-
ferent subpopulations of dermal DC (dDC) comprising a small
population of Langerin+ DC and a major heterogeneous pop-
ulation of Langerin− CD11blow/+ DC [130]. Langerin+ conven- 8.2 Materials
tional DC (cDC)1, identified by the expression of the chemokine
receptor XCR1 [131], can be further subdivided into CD103+ and 8.2.1 General reagents required for the crawl-out and FITC
CD103− subsets, which both display low expression of CD11b and painting assays
Epcam [130]. These Langerin+ cDC1 are critical to cross-present
exogenous Ag to CD8+ T cells promoting cytotoxicity [132], All necessary reagents that are needed for the crawl-out as well
however, cross-presentation capacity was also demonstrated for as for the FITC painting assay are listed in Table 22.
Langerin−CD11blow cDC2 [133] and LC [134]. Langerin−CD11b+
cDC2 are also competent to prime cytotoxic T cells [135] as well 8.2.2 General solutions for the crawl-out and FITC painting
as efficient in presenting antigens on MHC class II to CD4+ T cells assays
[136].
Mobilization and migration are critical features of effective All necessary solutions that are needed for the crawl-out as well
LC and DC function and therefore important to study. Here, we as for the FITC painting assay are listed in Table 23.
describe two complementary protocols for the analysis of skin
DC migration: firstly, an ex vivo DC migration assay of mouse
ear skin explants (the so-called ‘crawl-out assay’) and secondly, 8.2.3 Reagents specific for the crawl-out assay
FITC-painting as an in vivo assay to monitor DC trafficking to skin-
draining LN in an inflammatory setting. Moreover, for both meth- All necessary reagents that are specifically needed for the crawl-
ods, we provide an easy and ready-to-use protocol for the flow out assay are listed in Table 24.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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Eur. J. Immunol. 2023;53:2249925 Analyzing mouse skin DC migration by whole skin explant culture and FITC painting assays 49 of 96
Table 25. Specific solutions for the crawl-out assay Table 28. General equipment for the crawl-out and the FITC painting
assay
Solution Ingredients End concentration
Equipment Manufacturer Catalogue
R10-medium RPMI (+ glutamine) 10% number
FCS 100 U/mL
Penicillin/Streptomycin Forceps & Scissors Sharp tissue scissors HSB-390-10
HSB Hammacher (51807020)
Falcon (50mL) Greiner bio-one 227261
Table 26. Specific reagents for the FITC painting assay 96-well plate (V shape) Thermo Scientific 163320
1.5 mL tubes Sarstedt AG 72.690.001
Reagent Manufacturer Catalogue 70 um cell strainer Falcon 10082019
number Neubauer chamber Superior Marienfeld
Microscope Olympus CK2
DMSO Sigma 67-68-5 Centrifuge “Z 446 K” Hermle Labor
Dibutyl phalate (DBP) Sigma D2270 Technik
Aceton ApliChem 131007.1211 PipetBoy Fisher Scientific
FITC (Fluoresence Sigma F7250 FACS “Canto II” BD
isothyocynate
isomer I)
Collagenase IV Worthington LS0004186 Table 29. Special equipment for the crawl-out assay
Deoxyribonuclease I Roche 10104159001 Equipment Manufacturer Catalogue
(DNaseI) number
Table 27. Specific solutions for the FITC painting assay Incubator (37°C, 5% Heraeus BBD6220
CO2)
Solution Ingredients End concentration 24-well plate Thermo Fisher 160007
Scientific Inc.
LN Digestion mix RPMI (+ glutamine)
Collagenase IV 200 U/mL Table 30. Special equipment for the FITC painting assay
DNase I 0.5 U/mL
Equipment Manufacturer Catalogue
number
8.2.4 Solutions specific for the crawl-out assay
Isoflurane Vaporizer UniVet Porta
All necessary solutions that are specifically needed for the crawl- Thermo shaker Laboratory Technology
out assay are listed in Table 25. Buddeberg
8.2.5 Reagents specific for the FITC painting assay
8.2.9 Equipment specific for the FITC painting assay
All necessary reagents that are specifically needed for the FITC
All necessary equipment that is specifically needed for the FITC
painting assay are listed in Table 26.
painting assay is listed in Table 30.
8.2.6 Solutions specific for the FITC painting assay
8.3 Step-by-step sample preparation for the crawl-out
All necessary solutions that are specifically needed for the FITC assay
painting assay are listed in Table 27.
8.3.1 Preparation of ear tissue
8.2.7 General equipment
1. Cut off the ears at the base of the mouse ears (Fig. 18A)
All necessary equipment that is needed for the crawl-out as well 2. Split the ears into dorsal and ventral halves with strong for-
as for the FITC painting assay is listed in Table 28. ceps (Fig. 18B)
3. Add 2mL of R10-medium into a 24-well plate
4. Place each of the four ear halves with the dermal side down
8.2.8 Equipment specific for the crawl-out assay onto medium in a 24-well plate (one ear per well, skin should
float on medium) (Fig. 18C)
All necessary equipment that is specifically needed for the crawl- 5. Culture ear skin for 24h or 48h in the incubator (37°C, 5%
out assay is listed in Table 29. CO2) to allow skin DC to emigrate from tissue
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50 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Figure 18. Illustration of the different steps of the crawl-out assay. Ears are cut off at the base of the mouse ear cartilage (A) and afterward split
with two fine forceps to separate ventral and dorsal sides from each other (B). Subsequently, each ear halve is placed with the dermal side down
into a separate well (24-well plate) filled with 2 mL medium (C).
8.3.2 Harvesting cells after 24h and 48h 4. Anaesthetize mice with Isoflurane (0.5 L/min. O2, 3 L/min.
Isoflurane)
All following steps can be performed under non-sterile condi- 5. Paint both ears with 25 μL FITC solution per ear (dorsal and
tions. ventral side) (Fig. 19A&B)
6. Keep mice anesthetized until FITC solution has dried on both
6. Put the 24-well plate containing ear skin explants on ice for ears
10 min. 7. Isolate the skin-draining cervical LN after 24h and 48h (Fig.
7. Remove skin from medium by pulling the dermal side over 19C)
the well edge to catch migratory DC stuck to the dermal side
and harvest the cells by transferring the culture medium into
Note: Isolate skin-draining inguinal LN not draining the FITC-
a 50 mL Falcon tube
painted site to serve as a negative control
8. Wash each well by adding 2 mL PBS/EDTA solution and incu-
bate the plate for 10 min. in the incubator (37°C, 5% CO2)
9. Collect leftover cells and add them into the 50 mL Falcon
tube 8.4.2 Single-cell suspension of lymph nodes
10. Centrifuge cells for 5 min. at 450 × g at 4°C
11. Resuspend the pellet in 500 μL of PBS/EDTA solution and All following steps can be performed under non-sterile condi-
count cells tions.
8.4 Step-by-step sample preparation for the FITC 1. Put both cervical LN in a 1.5 mL Eppendorf tube filled with
painting assay 10 μL PBS and check that both LN are covered in liquid (so
that they will not dry out)
8.4.1 Applying FITC reagent mix on mouse ear skin 2. Add about 200 μL of LN digestion mix
3. Cut the LN into very small pieces with a fine scissor
1. Dissolve FITC powder in DMSO (Table 31) 4. Add another 800 μL of digestion mix and shake the samples
2. Mix Aceton with DBP at a ratio of 1:1 at 1200 rpm for 45 min. at 37°C
3. Dilute FITC dissolved in DMSO 1:10 in Aceton/DBP and mix 5. Add 20 μL of 500 mM EDTA solution per 1 mL digestion mix
well (final concentration of EDTA in 1mL is 10 mM) for 5 min. at
room temperature
Table 31. List of different concentrations that can be used for the FITC 6. Vortex cell suspension and pass it over a 70 μM cell
painting assay strainer
7. Wash the 1.5 mL Eppendorf tube with 1 mL PBS/EDTA solu-
% FITC FITC per 100 μL DMSO
tion and also pass it over the 70 μM cell strainer
1% 1 mg 8. Wash the cell strainer with 10 mL PBS/EDTA solution
5% 5 mg 9. Centrifuge cells for 5 min. at 450 × g at 4°C
7.5% 7.5 mg 10. Resuspend the pellet in 1 mL PBS/EDTA solution and count
10% 10 mg cells
: FITC concentrations vary in the literature and should be deter- 11. Centrifuge cells for 5 min. at 450 × g at 4°CNote
mined on an individual basis; most commonly used concentrations 12. Resuspend the cell pellet in a volume of FACS buffer to reach
range from 1–5 % 2×106 cells/ 100 μL
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Figure 19. Experimental steps during FITC painting. Mice are anasthezised with Isoflurane (0.5 L/min O2, 3 L/min Isoflurane) and 25 μL of FITC
solution is equally distributed on the dorsal and ventral side of each ear (A). After 24h and 48h of FITC painting, ears of mice appear slightly yellow
(B). At these time points, the cervical lymph nodes are swollen and also appear slightly yellow (for anatomical orientation the trachea is indicated
with a black arrow) (C).
Table 32. List of surface staining antibodies including the used fluorochrome, company, clone, and dilution
Fluorochrome Surface molecule Company Clone Dilution
FITC* CD24 (optional) Biolegend M1/69 1:200
APC-Cy7 CD11c Biolegend N418 1:200
PE CD11b eBioscience M1/70 1:1000
PE-Cy7 CD326 (Epcam) eBioscience G8.8 1:800
ef506 Fixation Viability dyes eBioscience – 1:1000
PerCp-Cy 5.5 CD103 Biolegend 2E7 1:100
ef450 MHCII (I-A/I-E) eBioscience M5/114.15.2 1:4000
* Note for FITC painting: Leave the FITC channel empty without any staining antibody to detect the FITC+ DC that have migrated to the LN
8.5 General flow cytometry staining 11. Stain for intracellular molecules or molecules that need to be
stained with a preceding membrane permeabilization step to
All following steps can be performed under non-sterile condi- improve staining intensity (here: Langerin, CD207) in 50 μL
tions. BD Perm/Wash buffer for at least 30 min. or overnight in the
dark at 4°C (Table 33)
12. Add 100 μL BD Perm/Wash buffer and centrifuge for 5 min.
1. Take all the cells from one mouse (from four ear halves) for at 450 × g at 4°C
one flow cytometry staining 13. For washing the cells resuspend the cell pellet in 100 μl BD
2. Pipet cells into a V-bottom 96-well plate Perm/Wash buffer and centrifuge for 5 min. at 450 × g at 4°C
3. Commercially available, purified rat anti-mouse anti- 14. Wash the cells again with 100 μL FACS buffer and centrifuge
CD16/32 is employed to block the Fc-gamma RIIB/III recep- for 5 min. at 450 × g at 4°C
tors, thereby avoiding unspecific recognition of staining anti- 15. Resuspend the cell pellet in 100 μL FACS buffer
bodies by csurface Fc-gamma receptors. For this, add 10 μl 16. Cells are ready for flow cytometry analysis
of Fc solution to the cells and incubate for 15 min. at room
temperature and centrifuge cells for 5 min. at 450 × g at 4°C
4. Resuspend cells in 50 μL cell surface marker antibody master
mix (Table 32) in FACS buffer and incubate in the dark at 4°C 8.6 Data analysis & gating strategy of migrated ear DC
for at least 20 min.
5. Add 100 μL FACS buffer to each well to stop the staining Data acquisition of the samples was performed at a BD Canto
6. Centrifuge cells for 5 min. at 450 × g at 4°C II analyzer with a blue (488 nm), a violet (405 nm), and red
7. Add again 100 μL FACS buffer and resuspend the cell pellet (633 nm) laser. Possible channels (and their filters) that can be
8. Centrifuge cells again for 5 min. at 450 × g at 4°C used are: FITC (530/30 nm), PE (564/42 nm), PerCp (>670
9. Fix the cells by resuspending the cell pellet in 100 μL of BD nm), PeCy7 (780/60 nm), APC (660/20 nm), APC-Cy7 (780/60),
Cyofix/Cytoperm solution for 45 min. in the dark at room Pacific blue (450/40), and Amcyan (530/70 nm). Data were ana-
temperature lyzed by FlowJo software (V10.8.0). In the following, an example
10. Add 100 μL of 1:10 diluted (in H2O) BD Perm/Wash buffer of a gating strategy for crawl-out DC (24h and 48h, Fig. 20) and
and centrifuge for 5 min. at 450 × g at 4°C FITC+ LN DC (24h and 48h, Fig. 21) is provided:
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52 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 33. List of molecules that need to be stained after membrane permeabilization including the used fluorochrome, company, clone and
dilution
Fluorochrome Surface molecule Company Clone Dilution
AF647 CD207 (Langerin) Dendritics 929F3.01 1:200
8.7 Pitfalls and top tricks immune cells by using a leukocyte marker, such as the leuko-
cyte common antigen CD45.
• Crawl-out assay: It is possible to digest the ear skin to charac- • FITC-painting: Always take a skin-draining LN as a negative
terize the ‘left over’ DC in the skin after 24h or 48h (digestion control to facilitate the setting of the right gate for the FITC-
protocol for ears see section 1.1. of Guidelines for DC prepa- signal during flow cytometry analysis. This can be either an ear
ration and flow cytometry analysis of mouse non-lymphoid skin-draining LN from an untreated mouse or a skin-draining
tissues [137]). The skin is a non-lymphoid tissue and harbors LN of a FITC-treated mouse distant from the ears (e.g. an
not only leukocytes. Therefore, it is recommended to identify inguinal LN).
Figure 20. Example gating strategy for flow cytometry analysis of crawl-out DC after 24h and 48h incubation. Cells emigrated from ear skin
explants were stained and first analyzed by FSC-A and SSC-A to exclude debris, followed by elimination of doublets. DC are identified as living
MHCII+CD11c+ cells (A), and gated into the different DC subsets at 24 h (B) and 48 h (C) of explant culture. Langerhans cells (LC) and dermal DC
(dDC) subsets were categorized by their expression of Langerin and EpCam. LC are double-positive for EpCam and Langerin, Langerin+ dDC are
only positive for Langerin and Langerin– dDC are negative for both markers. Langerin+ dDC were further divided into CD103– and CD103+ subsets.
Furthermore, CD11b+ dDC are identified as CD11b+Langerin– EpCam– DC.
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Eur. J. Immunol. 2023;53:2249925 Analyzing mouse skin DC migration by whole skin explant culture and FITC painting assays 53 of 96
Figure 21. Example gating strategy for flow cytometry analysis of FITC+ DC 24h and 48h after FITC painting. Single cells of skin-draining LN were
stained and analyzed by FSC-A and SSC-A to exclude debris, followed by elimination of doublets. DC are identified as living MHCII+CD11c+ cells.
Among these cells, migratory DC can be distinguished from their LN-resident counterparts by higher expression of MHCII and lower expression of
CD11c (A). The frequency of FITC+ DC within the migratory DC population was determined by gating on a FITC-negative control (see Notes below)
(B). Subsequently, migratory FITC+ DC were divided into the distinct subsets 24h (C) and 48h (D) after FITC painting. Langerhans cells (LC) and
dermal DC (dDC) subsets were categorized by their expression of Langerin and EpCam. LC are double-positive for EpCam and Langerin, Langerin+
dDC are only positive for Langerin and Langerin– dDC are negative for both markers. Langerin+ dDC were further divided into CD103– and CD103+
subsets. Furthermore, CD11b+ dDC are identified as CD11b+Langerin– EpCam– DC.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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54 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
• Induction of TSLP secretion after FITC painting: DBP in the is the NLR family pyrin domain containing 3 (NLRP3) inflamma-
FITC-solution triggers thymic stromal lymphopoietin (TSLP) some, which recognizes a diverse set of ligands including danger-
secretion by keratinocytes and therefore this assay represents associated molecular patterns derived from host cells (e.g., intra-
an example of Th2-driven inflammation [138]. cellular stress and damage) as well as pathogen-associated molec-
• TRITC as an alternative for FITC: Using FITC as a cell tracker ular patterns deriving from bacteria or viruses [142, 147]. Besides
for the observation of in vivo skin DC migration can be critical their broad proinflammatory functions, both IL-18 as well as IL-
when working with transgenic GFP reporter mice. In this case, 1β are important for the polarization of CD4+ T cells into Th1
TRITC (red) can be used instead of FITC (green) to monitor and Th17 cells, respectively [148–150]. Thereby, activation of the
sdLN migration [139]. inflammasome might contribute to protective immune responses
• Staining of CD24 in skin and skin-draining LN: In the skin, it against invading pathogens [142, 151]. However, overt activation
is possible to use CD24 instead of Langerin (CD207) to dis- of the inflammasome is associated with different chronic inflam-
tinguish Langerin+ dermal DC as well as LC. This has the matory diseases such as rheumatoid arthritis [152].
advantage that only surface staining is needed and cells do The capacity of DC to activate the inflammasome and secrete
not need to be permeabilized. Notably, the expression of CD24 IL-1β is controversially discussed [142]. Most of the data were
is upregulated on DC during migration, thus it is not advis- generated using bone marrow- (mouse) or monocyte-derived
able to use CD24 in LN as a discriminating marker for CD24high (human) DC, while only few groups used bona fide primary
DC (which are also Langerin-positive) and CD24low DC (which DC: here, it is clearly shown that human primary cDC2 are able
are Langerin-negative and do not just represent migrated DC) to activate the NLRP3 inflammasome, while it seems that in
[130]. mouse DC activation is partially inhibited [142, 153–155]. In this
• Migration of LC: Once LC have been activated, they downreg- section, we provide a protocol to measure inflammasome activity
ulate EpCam expression to gain mobility and emigrate from the on different level (i.e., secretion of inflammasome-dependent
epidermis [140]. Special about LC is that their migration to the cytokines, inflammasome-induced cell death (pyroptosis), activa-
skin-draining LN occurs in two steps: first, LC migrate from the tion of the effector caspase-1) for cell sorted primary human DC
epidermis to the dermis across the basement membrane at the [153].
dermal-epidermal junction and then they traffic from the der-
mis to the draining LN with the afferent lymph [141].
• Kinetics of DC subset migration: In contrast to dermal DC, 9.2 Materials
LC need to pass the basement membrane at the dermal-
epidermal border to reach the draining lymphatics. There- 9.2.1 Reagents
fore, dermal DC and LC display different migration kinetics
especially detectable in the crawl-out assay. While the major- A complete list of reagents is provided in Table 34. Further, cell-
ity of dermal DC have emigrated 24h after activation, LC sorted primary DC are needed (see [156] section 3 “Cell sorting
are the main population of migratory DC appearing at 48h of primary human DC”).
(Fig. 20).
9.2.2 Equipment
9 Measuring inflammasome activity in
human primary DC Necessary equipment is listed in Table 35.
9.1 Introduction
9.3 Step-by-step sample preparation
Inflammasomes are multiprotein complexes in the cytosol that
are crucial for the activation of caspase-1 in order to cleave pro- 9.3.1 Preparation of stocks and solutions
IL-1β and pro-IL-18 into their mature form [142, 143]. Further,
caspase-1 cleaves the protein gasdermin D leading to the inser- DC medium
tion of gasdermin D into the cell membrane [144–146]. Thereby, Add 5% human sera type AB (v/v), 5% Panexin NTS (v/v),
gasdermin D forms pores enabling the diffusion of IL-1β and IL-18 5% Panexin NTA (v/v), 1% Penicillin-Streptomycin (v/v), 1% L-
into the extracellular space as well as inducing an inflammatory Glutamine (v/v), and 1% sodium pyruvate (v/v) to 500 ml RPMI-
form of cell death called pyroptosis. In human and mice, several 1640.
different inflammasomes exist, which mainly differ in the sen-
sor protein used for detecting danger and pathogen-associated ATP
signals [142, 143]. They however all use the protein apoptosis- Prepare a 200 mM stock solution of ATP by adding 9.072
associated speck-like protein containing a CARD (ASC) for multi- ml of endotoxin-free water to 1 g of ATP. Vortex the solution
merization and recruitment of pro-caspase-1, which subsequently until it is completely dissolved. Prepare aliquots and store them
activates itself by autoproteolysis. The best-studied inflammasome at –20°C.
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Table 34. List of needed reagents for the preparation of single cells Working solution for LDH-cytox assay
from human lymphoid tissues To prepare the Working Solution, add 5 ml volume of Assay Buffer
Reagent Manufacturer Ordering to the Dye Mixture vial. Close the cap and dissolve the contents
Number completely. Add back the whole volume of the mixture prepared
to the Assay Buffer bottle. Store the Working Solution at 4°C and
Chemicals & Solutions protected from light. The working solution is stable for up to 6
Dulbecco´s Phosphate Sigma-Aldrich D8537 months after mixing.
Buffered Saline without
calcium and magnesium
RPMI-1640 Medium Sigma-Aldrich R8758 FAM FLICA
Human Serum type AB (male) Sigma-Aldrich H4522 Prepare a 150x stock of FAM FLICA by reconstituting a vial of
Trypan blue Sigma-Aldrich T6146 FAM FLICA in 50 μl DMSO. Aliquot the stock and store it up to six
Adenosine 5’-triphosphate Invivogen tlrl-atpl months at –20°C.
disodium salt (ATP)
R848 (Resiquimod) Invivogen tlrl-r848
4´,6-diamidino-2-phenylindole Thermo 62247 9.3.2 Stimulation of the NLRP3 inflammasome in human
(DAPI) Scientific DC with ATP
Panexin NTS PAN Biotech P04-
95080P 9.3.2.1 Measuring of secreted IL-1β.
Panexin NTA PAN Biotech P04-95070
Penicillin-Streptomycin Sigma-Aldrich P4333 9.3.2.1.1 Stimulation of the NLRP3 inflammasome.
L-Glutamine Sigma-Aldrich G7513
Sodium Pyruvate Sigma-Aldrich S8636
1. Isolate primary human cDC1, cDC2, pDC, and monocytes as
Kits
described in section 3 “Cell sorting of primary human DC” in
LDH-CytoxTM Assay Kit BioLegend 426401
FAM FLICATM Caspase-1 Kit Bio-Rad ICT097 [156]. Resuspend the isolated DC in DC medium to a final
5
LEGENDplex Human Cytokine BioLegend 740102 concentration of 4×10 cells/ml.
2 Panel 2. Prepare a 10 μg/ml solution of R848 by diluting R848 stock
solution 1:100 in DC medium. Dilute the 200 mM stock solu-
tion of ATP 1:40 in DC medium to reach a final concentration
R848 of 5 mM.
Prepare a 1 mg/ml stock solution of R848 by adding 500 μl of 3. Transfer 4×104 DC or monocytes (100 μl of the prepared sus-
endotoxin-free water to 500 μg of R848. Vortex the solution until pension in step 1) into a well of a sterile 96-well V-bottom
it is completely dissolved. Prepare aliquots and store them at plate. For each cell population to be analyzed, prepare four
–20°C. wells (see Table 36).
4. Mix two wells of the cells for each cell population 1:1 with DC
1× Wash buffer (LEGENDplex) medium (control without priming), while the other two wells
Dilute 2 ml of 20 x Wash buffer with 38 ml of H2O. for each cell population are diluted 1:1 with diluted R848
Table 35. Necessary equipment
Equipment Company Purpose
Centrifuge “Allegra X-15R” Beckman-Coulter Centrifugation of 50 ml tubes, 15 ml tubes, and V-bottom
plates
Incubator “HERAEUS BBD6220” Thermo Scientific Cabinet-style incubator with 5% CO2 and 96% relative
humidity for the lymphoid tissue digestion
LSR Fortessa (#647800) BD Flow cytometric analysis of single-cell suspensions
Neubauer chamber 0.100 mm; 0.0025 mm2 Superior Cell counting
Marienfeld
Sterile bench “Mars Safety Class 2” Scanlaf Performance of all aseptic procedures
96 Well Cell Culture Plate (V-bottom) (#651 Greiner bio-one Plates for the stimulation of sorted DC in sterile
180) conditions
Clear Flat-Bottom Immuno Non-sterile Thermo Scientific Performance of the LDH assay
96-Well Plates (#442404)
50 ml tubes (#352070) Falcon Preparation of 1× Wash buffer (LEGENDplex assay)
Serological pipettes (#606180) Greiner bio-one Pipetting
VersaMax enzyme-linked immunosorbent Molecular Devices Measuring of absorbance at 490 nm to detect cell lysis
assay plate reader
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56 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 36. Exemplary scheme for inflammasome stimulation for measuring of secreted IL-1β (9.3.2.1.)
Cell population Stimulation Well 1 Well 2 Well 3 Well 4
cDC1 1st (priming) DC medium DC medium 5 μg/ml R848 5 μg/ml R848
2nd (Inflam.) DC medium 5 mM ATP DC medium 5 mM ATP
cDC2 1st (priming) DC medium DC medium 5 μg/ml R848 5 μg/ml R848
2nd (Inflam.) DC medium 5 mM ATP DC medium 5 mM ATP
pDC 1st (priming) DC medium DC medium 5 μg/ml R848 5 μg/ml R848
2nd (Inflam.) DC medium 5 mM ATP DC medium 5 mM ATP
Monocytes 1st (priming) DC medium DC medium 5 μg/ml R848 5 μg/ml R848
2nd (Inflam.) DC medium 5 mM ATP DC medium 5 mM ATP
Inflam. = Inflammasome ligand
Table 37. Scheme for serial dilutions for the standard curve to measure secreted IL-1 β (9.3.2.1.)
C7 C6 C5 C4 C3 C2 C1 C0
Dilution Undiluted 1:4 1:16 1:64 1:256 1:1024 1:4096 blank
solution to reach a final concentration of 5 μg/ml R848 (see 11. Pipette 25 μl of assay buffer to 16 wells of a 96-well V-bottom
Table 36). Incubate the cells at 37°C for 3 h. plate for the standard curve (duplicates of each dilution and
5. Heat 10 ml of DC medium to 37°C. Preheat the prepared 5 mM two blanks). Further, add 25 μl of assay buffer to a well for
ATP solution. each sample that will be analyzed. For the scheme shown
6. After the 3 h incubation step, centrifuge the plate for 10 min above (Table 36), we would need 16 additional wells.
with 520 × g at RT. With a pipette, remove as much liquid as 12. Add 25 μl of the C1-C7 to the standard wells (duplicates).
possible without touching the cell pellet. Wash the cells with Add 25 μl of assay buffer to the remaining standard wells
100 μl of pre-warmed DC medium. Centrifuge the plate for (C0 = blank; as well in duplicates). Add 25 μl of the super-
10 min with 520 × g at RT. natants of the DC subpopulations and monocytes to the cor-
7. Remove the supernatant with a pipette and add 200 μl of responding wells on the 96-well plate.
prewarmed DC medium to one of the wells cultured in DC 13. Vortex the capture beads vial for at least 30 s. Then, add 25 μl
medium and one of the wells treated with R848. To the of the capture beads to each well of the standard curve (C0-
remaining wells (one cultured in DC medium and one treated C7) and the samples. Seal the plate with a provided plate
with R848) add 200 μl of the diluted ATP solution (see Table sealer and incubate on a plate shaker (800 rpm) in the dark
36). for 2 h.
8. Resuspend the cells and incubate for 3–12 h at 37°C. 14. Centrifuge the plate with 250 × g for 5 min at RT. Dump the
9. After the incubation time, centrifuge the plate for 10 min with supernatant into a sink and blot the plate on a stack of clean
520 × g at 4°C. Harvest the supernatants using a pipette and paper towel. Alternatively, remove as much liquid as possible
store either at –80°C or directly perform cytometric bead array with a pipette.
or ELISA to measure concentration of secreted cytokines (see 15. Wash the plate by dispensing 200 μL of 1 x Wash Buffer into
section 9.3.2.1.2.). each well and incubate for one minute. Repeat step 14.
16. Add 25 μL of Detection Antibodies to each well. Resuspend
the beads, seal the plate with a provided plate sealer and
9.3.2.1.2 Measuring cytokines in the supernatant of the cells. incubate the plate on a plate shaker (800 rpm) in the dark
for 1 h.
10. If supernatants were stored at –80°C, thaw the supernatants. 17. Without washing, add 25 μl of Streptavidin-PE to each well.
Warm up all contents of the LEGENDplex Human Cytokine Seal the plate with a provided plate sealer and incubate the
Panel 2 to RT before starting with the assay. Prepare a stock plate on a plate shaker (800 rpm) in the dark for 30 min
solution of the lyophilized cytokines for the standard curve by 18. Add 100 μl 1 x Wash Buffer to each well. Repeat steps 14 and
adding 250 μl of the assay buffer to the glass vial. Incubate it 15.
at RT for at least 15 min. Then, slightly pipette up and down. 19. Add 150 μl of 1 x Wash Buffer to each well and acquire
Prepare serial dilutions (1:4) by mixing 25 μl of the standard the samples on a flow cytometer. Cytometer setup can be
with 75 μl of assay buffer. Repeat this step five times by using performed using the cytometer setup beads provided by the
the diluted standard for the next dilution. In the end, one set. Follow the instructions in the manual of the manufac-
should have the top standard (C7) as well as six dilutions as turer. Briefly, record 300 events per bead. As we measure 13
shown in the scheme below (Table 37). cytokines, 3,900 events are recorded. Set the record/storage
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Table 38. Exemplary scheme for inflammasome stimulation for measuring of pyroptosis (9.3.2.2.),
Cells Well 1 high Well 2 low Well 3 Well 4 Well 5 Well 6
control control
cDC1 DC medium DC medium DC medium DC medium 5 μg/ml R848 5 μg/ml R848
DC medium DC medium DC medium 5 mM ATP DC medium 5 mM ATP
cDC2 DC medium DC medium DC medium DC medium 5 μg/ml R848 5 μg/ml R848
DC medium DC medium DC medium 5 mM ATP DC medium 5 mM ATP
pDC DC medium DC medium DC medium DC medium 5 μg/ml R848 5 μg/ml R848
DC medium DC medium DC medium 5 mM ATP DC medium 5 mM ATP
Monocytes DC medium DC medium DC medium DC medium 5 μg/ml R848 5 μg/ml R848
DC medium DC medium DC medium 5 mM ATP DC medium 5 mM ATP
gate to the bead population containing beads A and B so that 9.3.2.3 Measuring of active caspase-1.
only the beads are recorded and saved. This will simplify the
analysis using the LEGENDplex Software suite (see section 26. Isolate primary human cDC1, cDC2, pDC, and monocytes as
9.4.1). Adjust laser voltages for FITC, APC, and PE accord- described in section 3 “Cell sorting of primary human DC” in
ing to the manual and dependent on the used flow cytometer [156]. Resuspend the isolated DC in DC medium to a final
(for a LSRFortessa as used in this section, only APC and PE concentration of 1×106 cells/ml.
are needed). After cytometer setup, begin with sample acqui- 27. Transfer 1×105 DC or monocytes (100 μl of the prepared sus-
sition with the standard curve (C0-C7) starting with the low- pension in step 26) into a well of a sterile 96-well V-bottom
est concentration (C0). plate. For each cell population to be analyzed, prepare four
wells (see Table 36).
28. Stimulate the cells with R848 as described in 9.3.2.1. (step
9.3.2.2 Measuring of inflammasome-induced pyroptosis. 4). Incubate the plate at 37°C for 3 h. Shortly before the
end of the incubation time, prepare a 30× FLICA solution by
diluting the 150 x stock 1:5 in PBS. The diluted 30 x FLICA
20. Isolate primary human cDC1, cDC2, pDC, and monocytes as solution should be used within 30 min.
described in section 3 “Cell sorting of primary human DC” in 29. After washing as described in 9.3.2.1. (step 5–7), add 174 μl
[156]. Resuspend the isolated DC in DC medium to a final of the prepared 5 mM ATP solution or DC medium as con-
concentration of 2×106 cells/ml. trol as well as 6 μl of the 30× FLICA solution to each well.
21. Transfer 1×105 DC or monocytes (50 μl of the prepared sus- Resuspend the cells and incubate at 37°C for 3 h.
pension in step 20) into a well of a sterile 96-well V-bottom 30. Centrifuge the plate with 520 × g for 5 min at 4°C. Remove
plate. For each cell population, at least six wells are needed the supernatant and wash the cells with 150 μl of 1 x apop-
(see Table 38). tosis wash buffer.
22. Stimulate the NLRP3 inflammasome as described in 9.3.2.1. 31. Centrifuge the plate with 520 × g for 5 min at 4°C. Remove
(step 4–8). After addition of inflammasome stimulator ATP, the supernatant, add 150 μl of FACS buffer containing DAPI
incubate the plate at 37°C for 3–12 h dependent on the per- (1:10,000) and acquire the cells using a BD LSRFortessa.
formed assay. The volume in each well should be 50 μl.
23. 30 min before the incubation time ends, add 10 μl of Lysis
buffer (containing Octoxinol (Triton X-100); Lysis buffer is
part of the LDH-CytoxTM Assay Kit, BioLegend) to all high 9.4 Data analysis
control wells (Well 1 of Table 37) and 10 μl of DC medium
to all other wells. 9.4.1 Measuring of secreted IL-1β
24. Centrifuge the plate with 250 × g for 2 min at RT. Trans-
fer 50 μl of the supernatant into a new optically clear 96- 1. Data of experiment 9.3.2.1 are analysed using the
well F-bottom plate. Add 50 μl of working solution (contain- LEGENDplexTM Data Analysis Software Suite. In a first step,
ing 2H-Tetrazolium, 5-(2,4-disulfophenyl)-2-(4-iodophenyl)- acquired beads are separated into Beads A and B dependent
3-(4-nitrophenyl)-, inner salt, sodium salt (1:1); working on size and granularity (FSC-A vs. SSC-A) (Fig. 22A).
solution is part of the LDH-CytoxTM Assay Kit, BioLegend) 2. Then, individual beads are identified by APC signal as the dif-
to each well. Incubate for 30 min protected from light. ferent beads differ in the fluorescence intensity (Fig. 22A).
25. After the incubation time, add 25 μl of Stop solution (Stop Thereby, up to seven beads targeting different cytokines can
solution is part of the LDH-CytoxTM Assay Kit, BioLegend) to be measured for both Beads A and B.
each well. Measure the absorbance at 490 nm using an ELISA 3. On each gated bead, the fluorescence intensity in the PE chan-
reader. nel is measured (Fig. 22B). By generating a standard curve
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Figure 22. Measuring of secreted cytokines by ATP-treated human cDC2. Sorted human cDC2 were primed or not with 5 μg/ml R848 for 3 h. Then,
the inflammasome was activated or not with 5 mM ATP for 12 h. Subsequently, supernatants were harvested and stored at –80°C until analysis
using the LEGENDplex Human Cytokine 2 Panel. 4,000 beads per sample were acquired using an LSRFortessa. (A) Beads are separated into Beads
A and B using FSC-A/SSC-A. Then, each bead population is analyzed for signals in the APC channel and beads are divided into six (Beads A) or
seven (Beads) individual bead populations binding a specific cytokine. (B) Exemplary histogram gates for beads binding IL-1β, IL-23, or IL-18. Overlay
histograms (half-offset) show different experimental conditions. (C) Bar graphs show mean + SD of the concentration in the supernatant for IL-1β,
IL-23, and IL-18 of four healthy blood donors.
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Table 39. Summary of the results of the different experiments
Read out of the assays
Experimental condition 9.3.2.1 Cytokine secretion 9.3.2.2 LDH release 9.3.2.3 activation of
caspase-1
Medium only No cytokine secretion No cell-death induced No activation of caspase-1
release of LDH
Priming with R848 Secretion of inflammatory No cell-death induced No activation of caspase-1
cytokines with low levels of release of LDH
IL-1β
Priming with R848 and Secretion of High release of LDH due to Cells contain active
stimulation of NLRP3 inflammasome-dependent pyroptosis caspase-1 and undergo
with ATP cytokines pyroptosis
using C0-C7, the concentration of the cytokines for each sam- 9.4.3 Measuring of active caspase-1
ple is calculated (Fig. 22C).
4. In this experiment, cell-sorted cDC2 were stimulated as 8. FLICA is cell-permeant and will efficiently diffuse in and out
described above with R848 and the NLRP3 inflammasome of all cells. If there is active caspase-1 inside the cell, it will
activated with ATP. While the combination of R848 as priming covalently bind with FLICA and retain the green fluorescent
signal and ATP as inflammasome ligand induce the secretion signal within the cell. Unbound FLICA will diffuse out of the
of the highest amounts of the inflammasome-dependent IL-1β cell during the washing steps. Pyroptotic cells will retain a
and IL-18, the secretion of IL-23, which is induced by R848, higher concentration of FLICA and fluoresce brighter than
is lost (Fig. 22B-C; Table 39). healthy cells.
9. To further characterize the status of the cells, a live/dead
staining was performed using DAPI. As DAPI is not cell-
9.4.2 Measuring of inflammasome-induced pyroptosis permeable, it is excluded from living cells, while it can bind
to the DNA of cells with membrane pores or ruptures as it
5. Data of the experiment 9.3.2.2 are analyzed using Excel. Dur- typically occurs during pyroptosis. Therefore, the acquired
ing inflammatory cell death such as pyroptosis, lactate dehy-
drogenase (LDH), which is usually in the cytosol of a cell, is
released into the supernatant due to rupture of the membrane.
The LDH in the supernatant then catalyses dehydrogenation of
lactate to pyruvate thereby reducing NAD+ to NADH. NADH
reduces water-soluble tetrazolium salt in the presence of an
electron mediator to produce an orange formazan dye. The
absorbance (OD) of the orange formazan dye is measured and
is proportional to the released LDH. Thereby, cytotoxicity is
measured.
6. In order to determine how much cell death occurred, posi-
tive (high control) and negative (low control) controls have to
be performed and are used for the calculation of induced cell
death using this formula:
Cytotoxicity (%)
= OD (sample)− OD (low control) × 100
OD (high control)− OD (low control)
7. In this experiment, cell-sorted cDC2 were stimulated as
described above with R848 and the NLRP3 inflammasome lig- Figure 23. Measuring of ATP-induced cell death using an LDH-
cytotoxicity assay.Sorted human cDC2were primed or not with 5 μg/ml
and ATP. While medium or stimulation with R848 alone did R848 for 3 h. Then, the inflammasome was activated or not with 5 nM
not induce measurable cell death, the NLRP3 inflammasome ATP for 12 h. Subsequently, supernatants were harvested and analyzed
ligand ATP induced pyroptosis in almost all cells irrespective using the LDH-cytox Assay Kit. Absorbance (OD) was measured using a
VersaMax ELISA plate reader (Molecular Devices) and cytotoxicity deter-
of priming with R848 or not (Fig. 23; Table 39). mined using the formula described in 9.4.2. (step 6). Bar graphs show
mean + SD of five healthy blood donors.
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Figure 24. Detection of active caspase-1 in ATP-treated human cDC2. Sorted human cDC2 were primed or not with 5 μg/ml R848 for 3 h. Then, the
inflammasome was activated or not with 5 nM ATP for 3 h. Subsequently, cells were stained with DAPI (1:10,000) and samples were acquired using
an LSRFortessa and data analyzed using FlowJo software. (A) Pseudocolor dot plots show fluorescence signals in the FLICA and DAPI channels for
the different experimental conditions. Cells are divided into FLICA−DAPI−, FLICA+DAPI−, FLICA+DAPI+, and FLICA−DAPI+ cells using a quadrant
gate. (B) Bar graphs show mean + SD of the ratio of FLICA−DAPI−, FLICA+DAPI−, FLICA+DAPI+, and FLICA−DAPI+ cells of four healthy blood donors
as described in (A).
cells of assay 9.3.2.3. are analyzed using FlowJo software for Table 39). Also here, almost no cells died without activation
the fluorescence intensity of FLICA and DAPI. After a mor- of caspase-1 (FLICA−DAPI+).
phology gate to exclude debris and selection of single cells
using FSC-A vs. FSC-H, the remaining cells are analyzed for
FLICA and DAPI. Using a quadrant gate, cells are divided into 9.5 Pitfalls
(Fig. 24A):
◦ FLICA−DAPI− cells, which are alive and did not activate Problem: Discrimination of bead populations is not possible
caspase-1, (9.4.1)
◦ FLICA+DAPI− cells, which activated caspase-1 but are not Potential solutions:
dead yet, To discriminate 13 beads populations based on FSC-A, SSC-A and
◦ FLICA+DAPI+ cells, which activated caspase-1 and are APC signal, appropriate setup of the flow cytometer is very impor-
already pyroptotic, and tant. The setup for different flow cytometers, dependent on the
◦ FLICA−DAPI+ cells, which died but did not activate laser equipment, is explained in the manufacturer instructions
caspase-1 before. and should be performed as described. FSC-A and SSC-A have to
10. Sorted cDC2, which were cultured in medium or stimulated be adjusted so that two clearly separated bead populations (see
with R848 only, did neither show staining for FLICA nor DAPI Fig. 22A) are visible. Using the included beads for setup of the
(Fig. 24A-B; Table 39). Thus, the cells were alive and did flow cytometer, the APC signal should be adjusted in a way that
not activate the inflammasome. However, after addition of the beads with the lowest fluorescence have a signal of 1×102
ATP, the majority of the cells activated caspase-1 as measured and the beads with the strongest fluorescence should not exceed
by fluorescence in the FLICA channel. Further, half of the a signal of 5×104 to ensure proper gating using the software. In
FLICA+ cells were already pyroptotic (FLICA+DAPI+) as they case of problems with the separation of the beads, unused capture
stained brightly for DAPI in addition to FLICA (Fig. 24A-B; beads (see step 13 of section 9.3.2.1.2) should be acquired. If the
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Eur. J. Immunol. 2023;53:2249925 Measuring inflammasome activity in human primary DC 61 of 96
separation of all 13 bead populations is not possible, adjust either type. The protocol in this section works well for primary human
FSC-A/SSC-A to better segregate beads A from beads B (Fig. 22A, DC subpopulations but has to be adapted if monocyte-derived DC
left panel) or adjust APC to improve the space between the single are used. Here, the cell number in each well should be reduced to
beads (Fig. 22A, right panel). avoid too high values.
Problem: The LEGENDplex software does not calculate the Problem: ATP- or other inflammasome-stimulated samples do
standard curve (9.4.1) not contain any events (9.4.3)
Potential solutions: Potential solutions:
Different issues can lead to problems with the calculation of the If only the samples, which were stimulated with an inflamma-
standard curve, such as the reconstitution of the top standard some ligand, did not contain any events, then pyroptosis might
(C7), the serial dilution (C6-C1) as well as the setup of the flow have occurred too fast. Repeat the experiments with shorter incu-
cytometer: bation times for priming (e.g., only 2 h R848; step 28 of section
Reconstitution of the top standard (C7): Make sure that the 9.3.2.3) and inflammasome stimulation (e.g., only 1h ATP; step
lyophilised cytokines are properly reconstituted. Pre-heat the 29 of section 9.3.2.3).
lyophilised cytokines and the Assay buffer to RT. Add the Assay
buffer to the lyophilised cytokines without vortexing and do not Problem: No difference between inflammasome-stimulated
directly mix by pipetting up and down. Let it stand for at least and control-treated samples
10 min before gently mixing using a pipette. Potential solutions:
Serial dilution: For the standard curve, the top standard (C7) In case both control-treated as well as inflammasome-stimulated
has to be diluted serially 1:4 by mixing 75 μl of Assay buffer with DC show high cell death in the culture, culture conditions might
25 μl of standard. After addition of 25 μl of the top standard (C7) not be ideal for the cells. As LDH is released into the cell culture
to the 75 μl of Assay buffer (C6), mix the liquid in tube C6 prop- medium by different ways of cell death (e.g. necrosis and sec-
erly by pipetting up and down several times with an appropriate ondary apoptosis), cell culture conditions have to be optimized
pipette. As the tube contains 100 μl set a pipette to at least 75 μl to avoid any cell death due to the culture of the cells in plates.
to entirely mix it before removing 25 μl for the next dilution (C5). The used medium is optimized for human primary DC isolated
Proceed analogously until C1. Be aware that C0 is a blank. Do not from blood and might have to be adapted if other cells are used.
add 25 μl of C1 to C0. If working with monocyte-derived DC, the medium used for dif-
Setup of the flow cytometer: During setup of the flow cytome- ferentiation should be used for the assay. Further, only plates
ter for the acquisition of the samples, the PE channel, which is should be used that allow culture for at least 12h without induc-
used for the calculation of concentration of the cytokines, should tion of significant amounts of cell death. E.g., in case untreated
be adjusted to a mean value of 65 ± 5 for the gated bead pop- plastic dishes induce activation of the DC, this could lead to cell
ulation. Make sure that the setup beads as well as the unstained death with higher background values. Further, if cells are acti-
capture beads of the kit (see step 13 of section 9.3.2.1.2) are in the vated due to the culture condition, this will impair differences
range of 65 ± 5 or adjust the voltage for the PE channel accord- between primed and not primed samples as the activation might
ingly. After acquisition of the samples for the standard curve, do induce expression of inflammasome genes.
not directly throw away the tubes but keep them in case adjust-
ments to the PE channel have to be performed. If the top standard
during acquisition gives too strong signals in the PE channel (e.g., 9.6 Top tricks
reaches the end of the X-axis), the software will not calculate the
standard curve for these beads. Then, reduce the voltage for the This section describes the analysis of inflammasome responses in
PE channel even if it leads to lower mean values than 65 for the human DC after stimulation with ATP. However, as a vast array
unstained beads. Re-acquire few beads of C7 to assure that they of inflammasome-activating ligands exist and also certain viruses
do not touch the end of the axis anymore. Repeat the acquisition as well as bacteria activate different subtypes of the inflamma-
of all standard samples (C0-C7). Before the acquisition of all sam- some [142, 157–160], the protocol can be easily applied to other
ples, export the fcs files of the standard curve in order to test, stimuli (see for example [153]). In order to proof that inflam-
whether calculation of the standard curve works now. masomes are activated in response to distinct ligands, inhibition
of caspase-1 can be performed using ac-yvad-cmk (#inh-yvad,
Problem: The measured absorbance values are too low or too InvivoGen) [153]. Inhibition of caspase-1 should completely abol-
high (9.4.2) ish the secretion of IL-1β. In case markedly amounts of IL-1β are
Potential solutions: still detectable, non-canonical inflammasomes using other cas-
If the absorbance values in the high control are also too low or pases, such as caspase-4, -5 or -8, might be activated [161–167].
high, the number of cells used for the assay were inappropriate. In addition, specific inhibitors for NLRP3, such as MCC950 (#inh-
The absorbance is proportional to the released LDH. As dependent mcc, InvivoGen), exist, which can be used to test which inflamma-
on the cell size and the type of cell, the cytosolic amount of LDH some subtype is activated in response to newly identified or not
might vary, the cell number has to be adjusted to the used cell well-characterized inflammasome ligands [153].
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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62 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
In addition to human primary DC isolated from blood, the be provided to the T cells once the antigen is presented. There-
described assays can be performed with DC isolated from human fore, among all subtypes of APC DC are most efficient in express-
lymphoid tissues such as spleen. In case the DC are prematured ing a multiplicity of co-activation molecules such as CD80, CD86,
due to the isolation protocol, inflammasome activation with CD83, CD40, ICOS ligand [172]. Thus, with the right mixture of
subsequent pyroptosis and cytokine secretion might be observed MHC-II peptide complexes and T cell costimulatory molecules, DC
without an additional priming step. However, using our adjusted act as pivotal stimulators of an immune response.
protocols for isolation of lymphoid tissue DC (see section 1 Gen- However, this is a very sketchy plot of how antigen pre-
eration of single cell suspensions from lymphohematopoietic sentation by DC occurs, as the antigen presentation process
tissues in [156), inflammasome activation is not observed and the resulting T cell proliferation can be fine-tuned by sev-
without TLR-induced priming and subsequent inflammasome eral means. For instance, in activated DC antigen uptake (reg-
activation. ulated by differential receptor expression and/or re-direction of
intracellular receptor routing) and loading of MHCII molecules
(increased synthesis and enhanced loading/transport to the sur-
9.7 Summary table face) is effectively upregulated, leading to 10 to 1000-fold more
MHCII:peptide complexes reaching the cell surface as compared
This section describes assays to measure inflammasome activation to non–activated DC. To make regulation of the Immune response
in primary human dendritic cells using different readouts, such even more complex. DC are also capable of expression inhibitory
as release of inflammasome-dependent cytokines (9.3.2.1), LDH molecules such as PD-1 LAG3, BTLA4, which will counteract the
(9.3.2.2) or measuring active caspase-1 (9.3.2.3). The results for initial T cell activation guided by MHCII:peptide complexes. And
human cDC2 stimulated with the NLRP3 ligand ATP after priming of course, the secretion of pro- or anti-inflammatory cytokines by
with R848 are summarized in Table 39. differentially activated DC adds another facet to the regulatory
mechanisms of the DC-T cell interactions during antigen presen-
tation. Therefore, to test the antigen presentation capacity of DC
10 In vitro antigen-presentation assays
+ in vitro is a valid parameter to assess their physiologic functionwith murine DC and CD4 T cells that cannot be substituted by measuring surface molecule expres-
sion or cytokine secretion. In the following, we provide a protocol
10.1 Introduction using transgenic OTII mice to set up a non-radioactive in vitro
assay for DC assessment.
10.1.1 DC as antigen presenting cells
10.1.2 The choice of responder T cells
Dendritic cells (DC) are professional antigen-presenting cells
(APC) and are well equipped with MHC-class II molecules
Bulk T cells are used, i.e., we will not distinguish between naive
(MHCII) and T cell costimulatory molecules, such as CD80 and
and memory T cells. Of course, the animals are naive, however
CD86. In order to present antigens and to stimulate cognate
+ that does not mean that all T cells are truly naive as assessed byCD4 T cells, the first step is antigen uptake [168]. DC possess
being CD44 negative. Indeed, memory-phenotype T cells respond
three major possibilities to take up antigens: 1. by phagocytosis
to antigen in ways different from naïve-phenotype T cells [173].
(uptake of solid antigens), 2. by pinocytosis (“drinking”; uptake
Memory T cells proliferate in response to lower concentrations of
of antigens soluble in liquids), and 3. by absorptive endocyto-
antigen, do not require a long antigenic stimulation as compared
sis. That is, DC express defined surface receptors (see review
to naïve T cells, display effector functions (i.e., IL-2 secretion)
by Stoizner et al., this issue) that a capable to bind defined lig-
sooner after activation and they are less dependent on costimula-
ands and to mediate endocytosis and further intracellular rout-
+ tory signals than naïve T cells. Thus, in vivo activation of naïve Ting to MHCII compartments via coated vesicles. Following endo-
cells is strictly dependent on dendritic cells (DC), while memory
cytosis, ingested antigens have to access defined late endoso-
T cells also respond to antigen presented by other APC. But by
mal/lysosomal compartments where loading of MHC class II
using DC as stimulators in vitro, there are only marginal differ-
molecules occurs [169]. These endocytic compartments are also
ences in the proliferative response of naïve and memory-type T
referred to as MHCII compartments (MIICs) and are constitutively
cells, and the assay is suitable to generate a quantitative overview
synthesized by DC. In MIICs antigens are chopped into a variety
of the T cell stimulatory capacity of the DC subtypes used in the
of peptides of 13–25 amino acids in length and are bound to the
respective assays. Nevertheless, the researcher has to determine
binding-groove of newly synthesised MHCII in exchange for the
whether truly naïve T cell are necessary for the individual assay.
CLIP peptides that serve as final placeholder for antigenic pep-
tides in the fabrication process of MHCII. The now fully loaded
MHCII:peptide complexes are transported back to the surface and 10.1.3 The choice of viable or fixed DC
are now assessable for binding to cognate T cell receptors [170,
171]. Nevertheless, that alone is not sufficient to initiate T cell In the basic assay viable DC are used as stimulators, but as DC
proliferation, as T cell costimulatory signals (signal 2) have to as well as the responder T cells are viable, mutual interactions
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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Eur. J. Immunol. 2023;53:2249925 In vitro antigen-presentation assays with murine DC and CD4+ T cells 63 of 96
Table 40. Summary of standard chemicals for proliferation assays
Chemicals and Reagents Company Ordering number
Albumin Fraktion V, proteasefrei Carl Roth, Karlsruhe, Germany
CD4+ T cells Isolation Kit mouse Miltenyi Biotec, Bergisch Gladbach, Germany 130-104-454
eBioscienceTMCell Proliferation Dye Thermo Fisher Scientific, Dreieich, Germany 65-0842-85
eFluorTM 450
Fetal calf serum (FCS) Gibco, Darmstadt, Germany 10500-064
HEPES, ≥99.5% Carl Roth, Karlsruhe, Germany
L-Glutamine 200 mM Sigma, Taufkirchen, Germany
Minimum Essential Medium (MEM) Gibco, Darmstadt, Germany
OVA peptid (323-339) Genaxxon, Ulm, Germany P3287.9505
OVA protein Merk, Darmstadt, Germany
PBS Dulbecco w/o Ca2+ w/o Mg2+ Biochrom, Berlin, Germany
Penicillin/ Streptomycin (100×) Sigma, Taufkirchen, Germany
RPMI 1640 Bio&Sell, Feucht, Germany
Trypan Blue Stain, 0.4% Gibco, Darmstadt, Germany
Zombie AquaTM Fixable Viability Kit Biolegend, Koblenz, Germany 423101
Anti-CD3, Biotin Biolegend, Koblenz, Germany 100304
Anti CD28, Biotin Biolegend, Koblenz, Germany 102116
Anti CD4, PE Biolegend, Koblenz, Germany 100512
Table 41. Additional cell proliferation dyes
Cell tracker Company Ordering number Reference
CellTraceTM CFSE Cell Proliferation Kit Thermo Fisher Scientific, Dreieich, Germany C34554 [175]
CellTraceTM Violet cell proliferation kit Thermo Fisher Scientific, Dreieich, Germany C34557 [176]
Tag-It Violet Proliferation Cell Tracking Dye Biolegend, Koblenz, Germany 425101 [177]
Oregon Green 488, SE Thermo Fisher Scientific, Dreieich, Germany C34555 [178]
eBioscienceTM Cell Proliferation Dye Thermo Fisher Scientific, Dreieich, Germany 65-0840-90 [179]
eFluorTM 670
can take place and the phenotype of DC may change during cul- In Table 41 all cell proliferation dyes currently sold are listed.
ture. For instance, T cells can augment the expression of T-cell All were used according to the manufacturer’s protocols. So please
co-stimulatory molecules by DC, which in turn enables them to note to adjust cell density and washing steps.
stimulate proliferation of T cells rather antigen unspecific [174].
Moreover, different cytokine profiles of T cells (as a mixture of
naïve and memory like phenotype T cells may be used in the 10.2.2 Consumables
assays), may drive differentiation of DC in culture to a more or
less stimulatory phenotype. But as these effects may occur in par- All consumables are listed in Table 42. Some of the consumables
allel in all different test samples, the results obtained for T cell used for these experiments are sold from different vendors. It is
proliferation are valid. However, under certain circumstances it not mandatory to buy each part from the exact same vendor. For
may be important to freeze the status of the stimulatory DC to example, it is not necessary to buy all plastic ware from Greiner.
avoid any changes and interactions with T cells in vitro. To this
end DC can be fixed with paraformaldehyde (PFA) prior to setting 10.2.3 Devices
up the stimulatory assays. For that reason, a subsection of how to
fix DC is included into the protocol. The devices listed in Table 43 are produced from different com-
panies. It is not necessary to buy this equipment from the exact
10.2 Materials same vendor that is listed here. These are just examples from our
laboratory.
10.2.1 Chemicals and reagents
10.2.4 Buffers and solutions
Table 40 summarizes all chemicals used for the assays. They were
purchased as cell culture grade, if available. If not otherwise The buffers and solutions listed in Table 44 can be purchased as
stated the highest purity was obtained. ready to use solutions. However, for the experiments shown in
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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64 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 42. Standard equipment for proliferation assays
Consumables Company
Falcon 5ml Polystyrene Round-Bottom Tube Corning Lifescience, Amsterdam, Netherlands Corning
LifeScience, Amsterdam, derlandsNetherlandsork, USA
(12×75mm); FACS tubes
Costar 96 well cell culture round bottom plates well) Corning Lifescience, Amsterdam, Netherlands
Cell strainers (40 μm, 70 μm) Greiner, Frickenhausen, Germany
Magnetic LS Columns Miltenyi Biotec, Bergisch Gladbach, Germany
Conical tubes, 15 ml /50 ml Greiner, Frickenhausen, Germany
Sterile filter (0.2 μm) Sartorius, Göttingen, Germany
Pipette Tips (10 μl, 200 μl, 1000 μl) Greiner, Frickenhausen, Germany
Costar Pipettes (1 ml, 5 ml, 10 ml, 25 ml) Corning Lifescience, Amsterdam, Netherlands
Corning, New York, USA
this section, the buffers were prepared from solid chemicals. If 4. Centrifuge at 365×g for 7 min at 4°C (technical equipment,
not otherwise stated all solutions were sterile filtered and kept at ie. centrifuges etc are listed in Table 43).
4°C in the fridge for up to 4 weeks. 5. Discard the supernatant, resuspend cell pellets with 1ml
MACS buffer.
6. Fill up with 20 ml MACS buffer.
10.3 Step-by-step sample preparation 7. Centrifuge at 365 × g for 7 min at 4°C.
8. Discard the supernatant, resuspend cell pellets with 10 ml
MACS buffer.
10.3.1 Preparation of single-cell suspensions from lymph 9. Filter cells via a 70 μm cell strainer.
nodes and spleen 10. Count cells.
11. Fill up with 40 ml MACS buffer.
Note: You have you better yields when isolating spleen cells and 12. Centrifuge at 365 × g for 7 min at 4°C.
LN cells separately. 13. Discard supernatant, resuspend in MACS buffer to 1×107
cells per 40 μl MACS buffer.
10.3.1.1 Lymph nodes.
1. Sacrifice an OTII (B6.Cg-Tg(TcraTcrb)425Cbn/J) mouse and
10.3.1.2 Spleens.
collect lymph nodes (cervical, inguinal, axillary) into 10 ml
MEM medium (media and buffers are listed in Table 40 and
preparation is depicted in Table 44) in a 50 ml conical tube
(consumables are listed in Table 42). 1. Put spleens into 10 ml MEM medium in 50ml conical tube.
2. Grind through a 70 μm cell strainer. 2. Grind through a 70 μm cell strainer.
3. Fill up with 20 ml MEM medium. 3. Fill up with 40 ml MEM medium.
4. Centrifuge at 365 × g for 7 min at 4°C.
5. Discard the supernatant, resuspend cell pellets with 3 ml ACK
Table 43. Technical equipment for proliferation assays
Lysis buffer per spleen for 3 min at RT.
Device Company 6. Fill up with 40 ml MEM medium.
7. Centrifuge at 365g for 7min at 4°C.
Benchtop pH meters pH 50 Carl Roth, Karlsruhe, Germany 8. Discard the supernatant, resuspend cell pellets with 1 ml
VioLab MACS buffer.
Centrifuge Multifuge 3 S-R Thermo Fisher Scientific,
9. Fill up with 40 ml MACS buffer.
Dreieich, Germany
CO2 Incubator MCO-20AIC Sanyo Electric, Tokyo, Japan
10. Centrifuge at 365 × g for 7min at 4°C.
Spectrophotometer/ Germany 11. Discard the supernatant, resuspend cell pellets with 10 ml
Fluorometer MACS buffer per spleen.
Gallios flow cytometer Beckman Coulter, Krefeld, 12. Filter cells via a 70 μm cell strainer.
Germany 13. Count cell numbers.
MACS® MultiStand Miltenyi Biotec, Bergisch 14. Fill up with 40 ml MACS buffer.
Gladbach, Germany 15. Centrifuge at 365 × g for 7 min at 4°C.
Vortex Reax top Heidolph Instruments, 16. Discard the supernatant, cell pellets are single-cell suspen-
Schwabach, Germany sions.
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Eur. J. Immunol. 2023;53:2249925 In vitro antigen-presentation assays with murine DC and CD4+ T cells 65 of 96
Table 44. Preparation of buffers 4. Add 20μl anti-biotin microbeads per 107 cells (or scale up
accordingly).
Buffer Amount/ Ingredient
5. Mix well and incubate on the shaker for 15 min at 4°C.
Concentration
6. Fill up with 20 ml MACS buffer.
ACK lysis 1.658g NH4Cl 7. Centrifuge at 300 × g for 10min at 4°C.
buffer 8. Discard the supernatant, resuspend cell pellets in 5 ml MACS
0.2g KHCO3 buffer.
0.0076g EDTA 9. Place LS columns in the magnetic field of a suitable MACS
add to 0.20L ddH2O Separator.
(pH7.2)
× 10. Prepare column by rinsing with 3 ml MACS buffer.Complete 1 RPMI 1640
medium 11. Apply cell suspension onto the column.
1% L-Glutamine 200mM 12. Collect flow-through containing unlabeled cells, representing
+
1% Penicillin/ the enriched CD4 T cells.
Streptomycin 13. Wash column with 3ml MACS buffer.
(100×) 14. Collect unlabeled cells that pass through, representing the
10% FCS enriched CD4+ T cells.
1% HEPES (1M)
EDTA (100mM) 3.72g EDTA
add to 0.10L ddH2O 10.3.3 Labeling of Cell Proliferation Dye eFluorTM 450
(pH7.2)
FACS buffer 1× PBS
3% FCS Many protocols use CFSE labeling. That is still possible, however
HEPES (1M) 47.66g HEPES when using it side-by-side eFluorTM 450 we found it less toxic for
add to 0.20L ddH2O the cells and a more intensive stain (8 rounds of proliferation are
(pH7.2) clearly visible). Please note the materials section. Different dyes
MACS buffer 1× PBS (Table 41) are available, depending on your color-needs.
0.5% BSA
2mM EDTA 1. Wash cells two times with PBS to remove any serum. (Optional
MEM medium 47.65g MEM keep 1×104 cells in 300 μl FACS buffer in Facs tubes on ice as
9.25g NaHCO3 negative control).
23.85g HEPES 2. Resuspend cells at 2× the desired final concentration in PBS
1% Penicillin/ (pre-warmed to room temperature). For example, if the final
Streptomycin
× concentration of cells desired is 10×10
6/ml, then resuspend
(100 )
6
17.5 L 2-Mercaptoethanol cells at 20×10 /ml.μ
add to 5L (pH7.2) ddH O 3. Prepare a 20 μM solution of cell proliferation dye eFluor 4502
PBS (1× 5L) 47.75g PBS Dulbecco w/o in PBS (pre-warmed to room temperature).
Ca2+ w/o Mg2+ 4. While vortexing cells, add an equal volume of the 20 μM dye
add to 5.00L ddH2O solution prepared in step 4.
(pH7.2) 5. Incubate for 10 min at 37°C in the dark.
Trypsin lysis 1× PBS 6. Stop labeling by adding 4–5 volumes of cold complete media
buffer (containing 10% FCS) and incubate on ice for 5 min.
0.5% Trypsin 7. Wash cells 3 times with complete media. Count and check via-
5mM EDTA bility by trypan blue exclusion (viability >85%). (Optional:
Prepare approx. 1×104 cells/300 μl FACS buffer and test for
successful labeling. Negative cells (step 1) serve as controls).
10.3.2 Isolation of CD4+ T cells from single-cell suspensions
of spleens and lymph nodes (CD4+ T cells Isolation 10.3.4 Setting up T Cell proliferation assays
Kit, Miltenyi Biotec1)
10.3.4.1 Ova–pulsed DC.
1. Add 10 μl biotin-antibody cocktail per 40 μl of 107 cells. Use
more beads when you have more cells/volume but keep the 1. Stimulatory DC can either be in vitro prepared BMDC (see sec-
ratio of cells, buffer volume, and antibody cocktail. tion 7.3.1) or can directly be isolated from tissues (see section
2. Mix well and incubate on the shaker for 10min at 4°C. 8.3 and 8.4). DC have to be loaded with Ova peptide 323–
3. Resuspend cells with 30 μl MACS buffer per 107 cells (or 339 or with Ovalbumin protein. This can be used to eluci-
scale up accordingly). date the capability of DC to actively process antigen. When
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66 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
using Ovalbumin protein, DC need to uptake and to process it Add 100 μl/well of diluted antibody to the wells. Just add PBS
intracellularly, whereas Ova peptide can be presented to T cell to ‘non-activated T cells’. Incubate plate at 5% CO2 at 37°C for
without processing. Thus, a rough distinction between mature 2 hours.
(non-processing DC) and still premature (Ova protein process- 3. Retrieve 96-well plate from incubator after 2 hours. Aspirate
ing) DC can be made. Loading is achieved by adding Ova pep- the anti-CD3 and PBS solution and discard. Wash plate with
tide (concentration of 100 ng/ml) or Ova protein 100 μg/ml) 200 μl sterile cold PBS for each ‘activated T cells’ well. Finally,
to DC cultures for 6 h-12 h before harvesting. Of note, leave add 100 μl of complete medium to wells.
some aliquots of DC without Ova pulse. This will give you the
background proliferation of the T cells without cognate anti-
gen presentation. 10.3.4.3 Adding labeled T cells.
2. Harvest DC by careful pipetting (mature DC are sensitive to
shear forces) and wash 3 times with complete medium. Count
× 5 1. Dilute labeled T-cell suspension (see section 10.3.3) to a min-DC and suspend to a final volume of 4 10 cells/ml in com-
imum of 1×106/ml. 1×106 is rather the minimum. If avail-
plete medium.
able, we recommend to scale the number up to 1.2 – 1.3×106
2a OPTIONAL if fixed DC will be used in the assays: To fix DC
cell/ml. The proliferation is stronger.
4% PFA is used. Don’t use PFA solutions provided by several
2. Add 100 ul/well of the cell suspension to the DC plate. Pre-
companies, they are not pure enough and the pH is not correct.
pare one triplicate of “T cells” only and add 100 μl complete
Prepare 4% PFA solution as follows:
medium. Each well should now be filled with 200 μl medium.
1. Place 450 ml of dH2O in a glass beaker. Heat to 60°C using 3. Add 100 ul/well of the cell suspension to the plate labeled
a hot plate with stirring.
‘activated T cells’ and ‘non-activated T cells’. Into the ‘activated
2. While stirring, add 20 g of paraformaldehyde powder.
T cells’ wells, add (maximally 10 μl) anti-CD28 to a final con-
Cover and maintain at 60°C.
centration of 1 μg/ml.
3. Add 5 drops of 2N NaOH (1 drop per 100 ml). The solution
4. Incubate culture plate for 3–4 days in an incubator at 37°C,
should clear within a couple of minutes (There will be some
5% CO . Most T cells will require up to 3 days to divide. These
fine particles that will not go away). Do not heat solution 2
cells will aggregate but will not be attached to the plate.
above 70°C. PFA will break down at temperatures above
70°C.
4. Remove from heat and add 50 ml of 10× PBS. Adjust pH to
7.2; you may have to add some HCl. Adjust to final volume 10.3.5 Staining of CD4+ T cells and analysis by flow
of 500 ml. Use bottle top filter and place on ice. Cover with cytometry
foil to protect from light. Aliquots may be frozen at –20°C
and thawed as needed. 1. Harvest the T cell cultures by pipetting (200 μl) 2–3 wells to
5. Resuspend DC in PBS to 5×106 cells per ml PBS and add Polystyrene round-bottom tubes. Wash wells once with 200
an equal volume of 4% PFA to the tube. Mix (not vortex) μl of cold PBS and count cell numbers.
them and incubate at room temperature for 20 minutes 2. Wash cells with 3 ml PBS.
with intermittent shaking. 3. Dilute Zombie AquaTM dye for live/dead discrimination (exci-
6. Wash DC with complete medium 3 times and resuspend to tation by a violet laser) between 1:100-1:1000 in PBS. Resus-
concentrations (i.e. of 4×105 cells/ml) as needed for the pend 1–10×106 cells in 100 μl of the diluted Zombie AquaTM
assay. solution. To minimize background staining of live cells, titrate
3. Prefill as many rows as you need of a 96-well round bot- the amount of dye and/or number of cells per 100 μl for opti-
tom plate with complete medium. Add to the first three wells mal performance. Different cell types can have a wide degree
100 μl of the DC suspension (if possible, use multi-channel of variability in staining based on cell size and degree of cell
pipette). Make serial 1:2 dilutions by transferring 100 ul of death. In our cases, we dilute Zombie AquaTM dye at 1:500
the first 3 wells to the next. Mix the wells by pipetting 2-times in PBS and use 1×106 cells in 100 μl of a diluted Zombie
up and down and transfer again. Discard 100 ul from the last AquaTM solution. Incubate the cells at room temperature, in
3 wells. That way you will have triplicates of 2×104, 1×104, the dark, for 15–30 minutes.
5×103 and 2.5×103 DC per row. 4. Wash one time with 2 ml FACS buffer.
5. Stain 1×106 cells with anti-CD4/PE (clone RM4-5) (0.2μg)
10.3.4.2 T cell activation via anti-CD3 and anti-CD28 antibodies antibodies in 100μl FACS buffer.
(positive control). OPTIONAL: (1) Additionally, you can stain the DC in the cul-
ture by using ant-MHC class II antibodies in a suitable color.
1. Prepare 96-well round bottom plates by labeling at least 3 E.g., add 0.1ug anti MHC-II FITC or APC (clone M5/114)
wells with ‘activated T cells’ and ‘non-activated T cells’ together with anti CD4. You can “gate out” DC in your analy-
2. Coat the “activated T cell wells” with the anti-CD3 antibody sis. However, it’s not necessary as DC are „negative“ to „dim”
by diluting the anti-CD3 antibody to 10 μg/mL in sterile PBS. for CD4.
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Figure 25. Example for analyzing proliferation of T cells by FACS. CD4+ T cells pooled from spleen and lymph nodes were labeled with the pro-
liferation dye eFluor 450 and cultured with anti-CD3 and anti-CD28 antibodies for 4 days. Controls as indicated were cultured without antibody
stimulation. (A) Cells were stained with anti-Mouse CD4-PE and zombie aqua to discriminate live/dead cells and gated accordingly. (B) Viable
(ZOMBIE AQUA−) CD4+ cells were analyzed for eFluor F450 expression. Stimulated cells show a typical patterning indicating proliferation. In dou-
ble labeling experiments with CD73 APC one can clearly visualize that activated, proliferating cells downregulate CD73 during cycling. In any case,
unstimulated cells display no dilution of the dye. For further analysis individual eFluor 450+ peaks can be gated in histograms (C) and the percentage
of cells that had accomplished 1, 2, 3… rounds of proliferation can be depicted in a bar graph.
(2) Cells can easily be stained for other (activation) markers 10. Measure cells by Gallios flow cytometer (or alternative
of interest, i.e., CD25, CD69, CD73. That way one can inves- device).
tigate whether the respective maker is up-, or downregulated 11. Analyze data with Kaluza software (or alternative software).
during proliferation of the T cells. An example for downreg-
ulation of CD73 by activated/proliferated T cells is shown in
Fig. 25. 10.4 Data analysis
6. Incubate for 30 min at 4°C
7. Fill tube with 3 ml FACS buffer. Cells were analyzed with a Gallios flow cytometer and data were
8. Centrifuge at 365g for 7min at 4°C. visualized by Kaluza (BeckmanCoulter) software. In the Fig. 25
9. Discard the supernatant and resuspend cell pellets with 250 proliferation of CD4
+ T cells activated with anti-CD3 and anti-
μl FACS buffer. CD28 for 4 days is depicted. In case DC are used for stimulation,
a population of CD4− cells will appear left of the CD4+ gate.
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68 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
11 Quantitative analysis of in vitro 11.2 Materials
cross-presentation by mouse DC
11.2.1 Reagents
11.1 Introduction
A complete list of reagents is provided in Table 45.
An adaptive immune response is initiated when dendritic cells
(DC) recognize antigens in the peripheral tissue. Upon antigen
recognition and subsequent internalization, DC migrate towards 11.2.2 Equipment
the draining lymph nodes, where they can activate antigen-
specific T cells. For proper T cell activation, internalized anti- Necessary equipment is listed in Table 46.
gens must be processed intracellularly and antigen-derived pep-
tides are loaded onto major histocompatibility complex (MHC) 11.3 Step-by-step sample preparation
molecules. Peptides loaded on MHC II molecules can be recog-
nized by CD4+ T helper cells, whereas CD8+ cytotoxic T cells can
11.3.1 Preparation of stocks and solutions
be activated by peptides loaded on MHC I molecules. Presenta-
tion of extracellular antigens onto MHC I molecules is also termed
11.3.1.1 2mM EDTA solution.
cross-presentation [180].
Prepare a 0.5 M EDTA stock by dissolving 146.12 g EDTA in 800
Cross-presentation is mainly performed by cDC1 cells. It plays
ml bi-distilled water. Adjust the pH to 8.0 with NaOH to enable
an important role in the initiation of an immune response
dissolving. Fill up to 1000 ml with bi-distilled water and auto-
against viruses that do not infect DC, against tumor cells of non-
clave.
hematopoietic origin and in classical vaccination strategies aimed
Add 2 ml of the 0.5 M stock to 500 ml sterile DPBS to generate
at the induction of a potent cytotoxic T cell response. The molec-
2 mM EDTA/PBS.
ular mechanisms enabling cross-presentation, especially in vivo,
are not completely solved yet and are still topic of intensive inves-
tigations [180–182]. 11.3.1.2 mM β-Mercaptoethanol solution.
Antigen cross-presentation can be monitored directly by flow Prepare a 5 mM β-mercaptoethanol solution by diluting 174.8 μl
cytometry, using specific antibodies that recognize the complex β-mercaptoethanol in 500 ml DPBS and filter sterile.
between a loaded peptide and MHC I molecule, or indirectly
by measuring the proliferation or activation of antigen-specific
CD8+ T cells. Here, we will provide protocols for both direct and 11.3.1.3 Cell culture media.
indirect analysis of cross-presentation of the model antigen oval- For BM-DC medium, add 10% FBS Premium, 50 μM β-
bumin (OVA) in vitro. We will describe how cross-presentation mercaptoethanol, Penicillin/Streptomycin (10 units/ml, 0.1
of OVA can be measured directly using the 25-D1.16 antibody. mg/ml) as well as 2.5% J558 supernatant (final concentration
This antibody specifically recognizes the OVA epitope SIINFEKL 25 ng/ml) to IMDM medium. J558 supernatant serves as GM-CSF
(OVA257-264) when presented on the mouse MHC I in the context source for differentiation of the cells.
of H-2Kb [183]. Additionally, we describe how cross-presentation For T cell medium, add 10% FBS Standard, 50 μM β-
of OVA can be quantified after co-culture of OVA-loaded DC with mercaptoethanol and Penicillin/Streptomycin (10 units/ml, 0.1
OT-I cells, a specific CD8+ T cell clone that recognizes the same mg/ml) to RPMI 1640 medium.
OVA epitope on MHC I. After recognition of cross-presented OVA,
OT-I T cells become activated, proliferate, and secrete IL-2. Both 11.3.1.4 ELISA buffers.
proliferation and the secretion of IL-2 are proportional to the The ELISA wash buffer contains 0.05% Tween20 in DPBS.
amount of cross-presented OVA peptides and can be quantified To allow efficient immobilization of antibodies to positively
using flow cytometry or ELISA. To measure proliferation, T cells charged ELISA plates, a coating buffer with basic pH is required.
are incubated with carboxyfluorescein diacetate succinimidyl To prepare 1 l dissolve 8.401 g sodium bicarbonate in 950 ml bi-
ester (CFDA-SE) prior to their co-culture with antigen-treated distilled water and check the pH value. If necessary, adjust to pH
DC. CFDA-SE is internalized by the T cells and cleaved by 8.2. Fill the volume with bi-distilled water to 1 l and store at 4°C.
intracellular esterases into carboxyfluorescein succinimidyl ester For the ELISA blocking buffer add 1% BSA to DPBS.
(CFSE), which becomes membrane-impermeable and covalently 0.2 M sulfuric acid can be prepared by diluting 0.55 ml 18 M
links to cellular proteins. After each cell division, the fluorogenic sulfuric acid in 49.45 ml bi-distilled water.
CFSE is distributed among the daughter cells and fluorescence Dilute 1 mg NaPox in 200 μL bi-distilled water and fill up with
decreases accordingly. T cell activation can be easily monitored 800 μl DPBS to generate a stock solution of 1 mg/ml.
by determination of IL-2 in the supernatant of the cells using
ELISA.
Naturally, these protocols can be easily adapted for other anti- 11.3.1.5 FACS buffer.
gens / T cell clones. Add 0.5% BSA to DPBS.
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Eur. J. Immunol. 2023;53:2249925 Quantitative analysis of in vitro cross-presentation by mouse DC 69 of 96
Table 45. Reagents, enzymes, chemicals and solutions Table 45. (Continued)
Reagent Manufacturer Ordering Reagent Manufacturer Ordering
Number Number
Antibodies MS column Miltenyi Biotec 130-042-201
α-CD8-PerCP (53-6.7) ThermoScientific 46-0081-80 ELISA Plate F Form Greiner Bio-One 655061
α-CD11b-PE-Cy7 (M1/70) Invitrogen 25-0112-82 Cell scraper Sarstedt 83.3950
α-mouse IL-2 purified eBioscience 14-7022-85
(JES6-1A12) Table 46. Necessary equipment
α-mouse IL-2 eBioscience 13-7021-85
biotin-conjugated Equipment Company Purpose
(JES6-5H4)
α-SIINFEKL/H-2Kb clone Biolegend 141604 Scissors and Hartenstein Preparation of
25-D1.16-PE tweezers bones and
α-CD11c-eFluor450 eBioscience 48-0114-82 tissue from
Chemicals & Solutions mice
Mouse serum PAN-Biotech P30-0201M Microplate reader Tecan ELISA
PD10 desalting columns Merck GE17-0851-01 Infinite 200 PRO
Dulbecco’s Phosphate PAN Biotech P04-36500 BD LSR II Flow BD Biosciences Flow cytometry
Buffered Saline (DPBS, Cytometer
w/o Ca2+ and Mg2+)
RPMI 1640 medium PAN Biotech P04-22100 11.3.1.6 MACS buffer.
IMDM medium PAN Biotech P04-20150 MACS buffer contains 0.5% BSA and 2 mM EDTA in DPBS (pH
Penicillin-Streptomycin PAN Biotech P06-07100 7.2).
Fetal Bovine Serum (FBS) PAN Biotech P30-3306
Standard
Fetal Bovine Serum (FBS) PAN Biotech P30-1502 11.3.1.7 Purification of OVA.
Premium Before its application in cross-presentation experiments, we clear
β-Mercaptoethanol Roth 4227.3 OVA from small degradation products by gel filtration using PD-
Ethylendiamintetraacetic Roth CN06.2 10 Sephadex-25 desalting columns. By this means, one can avoid
acid (EDTA)
background signals due to alternative presentation or direct load-
OVA SERVA 11841.03
Trypan Blue Sigma T8154 ing of small peptide fragments.
CFSE Invitrogen 65-0850-85
1. Dissolve 300 mg OVA in 3 ml PBS in a 50 ml reaction tube by
OT I peptide (SIINFEKL) Anaspec 60193-1
Sodium bicarbonate Roth 965.2 gently swinging the reaction tube.
Tween20 Sigma Aldrich P1379-500ml 2. Centrifuge dissolved OVA 5 min at 3200 × g
DPBS powder (w/o Ca2+ PAN Biotech P04-36050P 3. Wash a PD-10 Desalting Column 5 times with 4 ml DPBS.
and Mg2+) Add the supernatant onto the column and discard the flow-
Albumin Fraction V (BSA) Roth 8076.4 through.
TMB One Kem-En-Tec 4395 4. Elute OVA from the column with 2.5 ml DPBS.
Mouse IL-2 recombinant eBioscience 148021-64 5. Determine OVA concentration at 280 nm with a UV-
protein Spectrophotometer.
Sulfuric acid (H2SO4) Honeywell Fluka 30743-1L-GL
CD8a (Ly-2) MicroBeads Miltenyi Biotec 130-117-044
mouse kit
NeutrAvidin Horseradish ThermoScientific A2664 11.3.2 Generation of bone marrow-derived DC (BM-DC)
Peroxidase conjugate
(NaPox) 1. Sacrifice C57BL/6J mice by cervical dislocation, fix at the
70 μm cell strainer Greiner Bio-One 542070 extremities and disinfect the ventral area of the chest,
40 μm cell strainer Greiner Bio-One 542040 abdomen, and hind legs.
5 mL syringe Braun 4616057V 2. Starting from the lower half of the abdomen, cut open the skin
10 mL syringe Braun 4616103V towards the chest and fix on each side. Starting from the same
Petri dish (10 cm) Sarstedt 82.1135.500 point, cut open the skin across both hind legs, exposing the
96 well plate Greiner Bio-One 655182 underlying tissues. After removing as much muscle and other
FACS Tubes Sarstedt 55.1579
× connective tissue from the legs as possible, proceed by dislo-Cannula sterican G26 1’’, Braun 4657683
× cating the femur from the pelvis and the tibia from the ankle,ø 0.25 40 mm
allowing excision of the leg bones without exposing the bone
(Continued) marrow to the unsterile environment. Submerge the leg bones
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70 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
in DPBS, then transfer to a DPBS-filled petri dish in a sterile 14. Add 50 μl antibody master mix per tube
environment for removal of any excess soft tissue either by 15. Incubate for 30 min at 4°C
cutting action or friction using a paper tissue. SeverSeparate 16. Wash cells with 2 ml FACS buffer, centrifuge for 5 min at
the femur from the tibia by overextension at the knee and cut 300 × g
open the resulting bone ends where necessary without sacri- 17. Collect cells in 300 μl FACS buffer for analysis by flow cytom-
ficing significant amounts of bone marrow. etry
3. Transfer the bones to a fresh DPBS-filled petri dish and fill a
10 ml syringe with DPBS. Using the syringe and a cannula
(G26), flush out all visible bone marrow, making sure to do so 11.3.4 Quantification of cross-presentation after co-culture
from both ends of each bone to maximize yield. Resuspend the with antigen-specific T cells by ELISA
cells, pass them through a 40 μm cell strainer, and centrifuge
at 300 × g for 5 min. Take up the cell pellet in warm BM-DC Stimulation of BM-DC with OVA or SIINFEKL
medium and plate the cells onto three 10 cm Petri dishes (12
ml per dish). Incubate the cells at 37°C and 5% CO2. 1. Harvest cells as in 3.3 using 2 mM EDTA
4. Split the cells from each dish onto two fresh dishes on days 3 2. Count the cells and resuspend them to a concentration of
or 4 post-isolation. To do so, collect the supernatant and incu- 2*106/ml in BM-DC medium. Plate 1*105 cells (50 μl) in a
bate the remaining adherent cells in 2mM EDTA solution at 96-well plate and incubate for at least 45 min at 37°C and 5%
37°C for 5 min. Wash the cells off the dish using a micropipette CO2.
and combine with the supernatant. Centrifuge the resulting 3. Stimulate cells with different OVA concentrations (0, 10, 100,
cell solution at 300 × g for 5 min, then aspirate the super- 200, 1000 μg/ml) or with the OT-I peptide (final concentra-
natant and take up the cell pellet in fresh BM-DC medium for tion 100 ng/ml). To do this, add another 50 μl medium with
plating. OVA or SIINFEKL in double concentration. Use freshly purified
5. The cells differentiate over the course of seven days, upon OVA as described in 11.3.1.7.
which they can be used for experimentation at a total yield 4. Incubate for 2 h at 37°C and 5% CO2.
of roughly 1.2-1.8×108 BM-DC per mouse.
Preparation of OT-I cells
11.3.3 Quantification of OVA cross-presentation by flow 1. Sacrifice an OT-I mouse by cervical dislocation and isolate the
cytometry using the 25-D1.16 antibody spleen. The following steps are performed under sterile condi-
tions.
For staining experiments, collect all BM-DC from one dish 2. Mash the spleen through a 70 μm cell strainer in a petri dish
filled with DPBS with the back of a 5 ml syringe.
1. Collect the supernatant of one petri dish in a 50 ml tube 3. Separate the cells by pipetting up and down and filter the sus-
2. Add 5 ml 2 mM EDTA solution to each plate, incubate for pension through a 40 μm cell strainer to remove remaining
5 min at 37°C clumps. Centrifuge the cell suspension at 450 × g for 10 min
3. Harvest cells from plate and add them to the supernatant and resuspend the cells in 10 ml T cell medium. Count cells
4. Centrifuge for 5 min at 300 × g and discard the supernatant and centrifuge again at 450 × g for 10 min at 4°C.
5. Resuspend the cells in BM-DC medium, count cells, and 4. CD8+ T cells were positively selected using the CD8a (Ly-2)
adjust the cells to 106 cells/ml MicroBeads mouse. Resuspend the cell pellet in 90 μl MACS
6. Plate 300 μl (300.000 cells) per well in a 24-well plate buffer per 107 cells and apply 10 μl of CD8a (Ly-2) MicroBeads
7. Incubate for at least 45 min at 37°C per 107 total cells. Mix and incubate the cells for 15 min
8. Add 100 μl medium containing OVA (final concentration of at 4°C.
5 mg/ml) or peptide (final concentration of 400 nM), incu- 5. Wash the cells with 2 ml of MACS buffer and centrifuge cells
bate for 6 h at 37°C at 300 × g for 10 min. Discard supernatant and resuspend the
9. Remove supernatant, add 500 μl 2mM EDTA solution per cell pellet in 500 μl MACS buffer.
well 6. Place the MS column into an appropriate separator and rinse
10. Harvest cells using a cell scraper the MS column with 500 μl MACS buffer. Transfer labeled cells
11. Collect cells in FACS tube, centrifuge for 5 min at 300 × g onto the column and discard flow-through.
12. Wash cells with 2 ml FACS buffer, centrifuge for 5 min at 7. Wash the column 3 times with 500 μl MACS buffer and discard
300 × g, discard the supernatant by decanting flow-through
13. Prepare antibody mastermix 8. Remove the column from the separator and elute cells with
FACS Buffer containing Mouse serum (1:32 diluted), 1 ml MACS buffer and a plunger. Collect flow-through and
fluorochrome-conjugated 25-D1.16 antibody (1:30 determine the cell number. Centrifuge at 300 × g for 10
diluted; final concentration 6 μg/ml), αCD11c (1:500 min. Adjust the cells to a concentration of 2*106/ml in T cell
diluted; final concentration 0.4 μg/ml) medium.
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Co-culture with T cells T cell isolation and CFSE staining
1. After incubation with OVA/SIINFEKL, remove the medium 1. Sacrifice an OT-I mouse by cervical dislocation and isolate the
from the BM-DC and add 2*105 purified T cells (100 μl) for spleen. The following steps are performed under sterile condi-
co-culture. tions.
2. After 18 h, transfer the supernatant onto a new 96-well plate 2. Mash the spleen through a 70 μm cell strainer in a petri dish
and store the plate at –80°C until further analysis by ELISA. filled with DPBS with the back of a 5 ml syringe.
3. Separate the cells by pipetting up and down and filter the sus-
ELISA pension through a 40 μm cell strainer to remove remaining
clumps. Centrifuge the cell suspension at 450 × g for 10 min
1. Coat a high-binding 96-well plate (ELISA plate) overnight and resuspend the cells in 10 ml DPBS. Count cells and cen-
(ON) with 50 μl/well of coating antibody (final concentra- trifuge again at 450 × g for 10 min at 4°C.
tion of 0.5 μg/ml) in coating buffer. Store the plate ON at 4. Add 10 ml DPBS with 2 μM CFSE and mix it with the cell
4°C. suspension (final CFSE concentration 1 μM).
2. After ON incubation, wash the ELISA plate 3 times with 150 5. Incubate for 15 min at 37°C in a 50 ml tube.
μl wash buffer. 6. Stop the reaction by adding 10 ml T cell medium and cen-
3. Add 100 μl/well blocking buffer and incubate for 1 h at RT. trifuge for 7 min at 450 × g.
4. Wash the plate 3 times with wash buffer. Ensure that no liquid 7. Resuspend the cells in 20 mL T cell medium and count the
remains before continuing cells. Adjust the cells to 1*106/ml in T cell medium.
5. Prepare a 1:2 dilution series of mouse IL-2 recombinant pro-
tein in blocking buffer, ranging from 0.12 ng/ml to 125
ng/ml. Load 50 μl sample and 50 μl of IL-2 standard range Co-culture with BM-DC
as well as medium as a negative control (Blank).
6. Incubate the plate for 2 h at RT and wash three times with 1. After 2 h of incubation with OVA or SIINFEKL, carefully
wash buffer. remove the medium from the BM-DC
7. Add 50 L/well biotinylated detection antibody (diluted 2. Add 1*105μ splenocytes (100 μl) for co-culture.
1:1000 in blocking buffer with a final concentration of 0.5 3. After 1 day, add additional 200 μl T cell medium to each well
μg/ml). Incubate for 1 h at RT. and incubate for 2 additional days.
8. Wash the plate thrice again in washing buffer.
9. Add 50 μl/well Neutravidin (diluted in PBS with a final con- Flow cytometry
centration 1 μg/ml) and incubate for 30 min at room tem-
perature. 1. After 3 days, transfer all cells into FACS tubes and add 2 ml
10. Wash 3 times and add 50 μl/well TMBOne substrate solution. DPBS to each tube. Centrifuge at 300× g for 5 min and discard
11. When the plate is sufficiently developed and a color change the supernatant by decanting.
can be seen, stop the reaction with 50 μl/well 0.2 M H2SO4. 2. Prepare antibody mastermix
12. Determine the optical density at 450 nm wavelength using a
DPBS containing mouse serum (1:100 diluted), fluorochrome-
spectrophotometer.
labeled antibodies against the surface antigens CD11b
(dilution 1:800; final concentration 0.25 μg/ml) and
CD8 (dilution 1:400; final concentration 0.5 μg/ml). also
11.3.5 Monitoring proliferation of activated T cells by flow include single stained samples as controls for proper gat-
cytometry ing.
3. Add 50 μl antibody master mix per tube
Stimulation of BM-DC with OVA or SIINFEKL
4. Incubate for 20 min at 4°C
5. Wash the cells again with 2 ml DPBS and take the cells up in
1. Harvest cells as in 3.3 using 2 mM EDTA
100 μl DPBS.
2. Count the cells and resuspend them to a concentration of
6 6. Analyze the cells by flow cytometry.1*10 /ml in BM-DC medium. Plate 5*104 cells (50 μl) in a
96-well plate and incubate for at least 45 min at 37°C and 5%
CO2.
3. Stimulate cells with different OVA concentrations (0, 100, 11.4 Data analysis
1000 μg/ml) or with the OT-I peptide (final concentration 100
ng/ml). To do this, add another 50 μl medium with OVA or For all flow cytometry experiments, a BD LSR II Flow Cytometer
SIINFEKL in double concentration. Use freshly purified OVA as was used and the data were analyzed using the FlowJo software.
described in 11.3.1.7. Spectrophotometrical analysis for ELISA was performed using a
4. Incubate for 2 h at 37°C and 5% CO2. Microplate reader Infinite 200 PRO (Tecan).
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Figure 26. Flow cytometric analysis of cross-presenting BM-DC using the 25-D1.16 antibody.
11.4.1 Analysis of cross-presentation using the 25-D1.16 11.4.3 Analysis of OT-I T cell proliferation by flow cytometry
antibody
CFSE profiles were depicted by flow cytometry as described above
Cells were gated as indicated. 25-D1.16 staining is shown for and shown in Fig. 28.
OVA- or SIINFEKL-treated BM-DC (Fig. 26). There are several ways to quantify proliferation data. Often,
BM-DC were treated with OVA or SIINFEKL and stained with the percentage of dividing cells is used for statistical analysis.
the 25-D1.16 antibody A) Exemplary gating of BM-DC. After elim- However, such analysis does not include the amount of cell
ination of doublets and debris, BM-DC are identified as CD11c+ cycles individual cells went through. Therefore, calculation of
cells. B) 25-D1.16 staining of OVA- or SIINFEKL-treated BM-DC
11.4.2 Analysis of OT-I T cell activation by ELISA
OD values were depicted by spectrometry as described above. Cal-
culation of IL-2 concentrations was done as follows using Graph-
padPrism
1. Subtract the OD of the blank from every other OD.
2. Transform the standard concentrations to logarithm of stan-
dard concentrations.
3. Plot the logarithm of concentration against the optical density
(Fig. 27A).
4. Perform a sigmoidal curve fitting with interpolation of Figure 27. IL-2 secretion by OT-I T cells is dependent on OVA concen-
unknown values from the standard curve. tration used for stimulation of BM-DC.A) Exemplary sigmoidal standard
curve. OD450 values of samples are shown in grey. B) IL-2 secretion by
5. Transform the logarithm of concentrations to concentrations OT-I T cells co-cultivated with BM-DC stimulated with various concen-
(x = 10×) trations of OVA or SIINFEKL peptide. Data are shown as mean ± SEM.
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Eur. J. Immunol. 2023;53:2249925 Quantitative analysis of in vitro cross-presentation by mouse DC 73 of 96
Figure 28. Flow cytometric analysis of T cell proliferation induced by OVA-stimulated BM-DC.
the division index is the scientifically more appropriate way to tively, it can be calculated with the proliferation tool of the FlowJo
quantify proliferation. software (see also 11.5.3.).
The division index is the average number of cell divisions that a Co-culture of CFSE-loaded OT-I T cells with OVA-stimulated
cell in the original population has undergone. It can be calculated BM-DC. A) Exemplary gating of T cells. After elimination of dou-
by the following formula: blets and debris, T cells are identified as CD8+ and CD11b− cells.
[ ] B) Proliferation profile of gated T cells. C) Division index of CD8+
· + · P1 + · P2 + · P3 + · P4 T cells co-cultured with BM-DC after OVA or SIINFEKL treatment.0 P0 1 2 2 4 3 8 4 16
+ · P5 + · P6 + · P7 Data are shown as mean ± SEM.5 32 6 64 7Division index = 128
+ · · ·
P0+ P1 + P2 + P3 + P4 + P52 4 8 16 32 + P6 P764 + 128 + · · ·
11.5 Pitfalls
with P0 the percentage of cells undergoing no division, P1 the per-
centage of cells undergoing one cell division, P2 the percentage of 11.5.1 Use of inhibitors/small molecules
cells undergoing 2 cell divisions,….
The division index can be calculated manually, setting gates When investigating the molecular mechanisms of cross-
for all populations with a specific number of cell cycles. Alterna- presentation, specific inhibitors/small molecules are often added
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74 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
to the DC culture. However, to avoid a direct influence of such 12 In vivo analysis of killing efficacy by
inhibitors on T cells, they need to be removed before the addition CD8+ T cell responses
of T cells. Consequently, if the inhibition is reversible, their effect
on DC would be lost during DC – T cell co-culture. To circumvent 12.1 Introduction
this problem, DC can be (mildly) fixed before the addition of T
cells. To do this, DC are washed with DPBS after incubation with Dendritic cells (DC) are important inducers of immune responses
the antigen. Afterward, DC are fixed with 0.008% glutaraldehyde to pathogens. This includes the priming of CD8+ cytotoxic T cells
for exactly 3 min and washed twice with DPBS again. Then, T needed to kill virus-infected and malignant cells [1]. Therefore,
cells can be added as described above. It is of particular impor- it is of utmost importance to monitor the functional capacity of
tance that the duration of this fixation step is identical for all the induced T cell responses. This allows assessment of the effi-
samples. cacy and efficiency of immunogenic therapies using DC as inducer
or mediator of anti-viral and/or antitumor responses without the
need to directly induce tumor formation or infect animals [3].
The here described protocol allows for the simultaneous measure-
11.5.2 Sensitivity of the 25-D1.16 antibody
ment of the killing efficacy of cytotoxic T cells to different peptides
within a single animal. Thereby, the numbers of animals needed
Since the sensitivity of the 25-D1.16 antibody is not very high, the
can be reduced and the T cells reactivity to different potential
signal is not easy to distinguish from background signal and, in
MHCI epitopes can be measured at the same time. The basis for
our hands, specific stainings are only possible using high amounts
the here presented protocol was published by Oehen et al., Quah
of antigen. For more sensitive assays with physiological antigen
et al., and Hermans et al. [184–186].
concentrations, T cell proliferation and IL-2 ELISAs should be
The principle of the assay is the differential labeling of the tar-
performed. In addition, we obtained best results using a directly
get cells and their loading with either different peptide concentra-
labeled antibody. In our hands, the use of a labeled secondary
tions and/or different peptides (different epitopes, irrelevant pep-
antibody also increased background staining, leading to a poor
tides). Therefore, splenocytes of a CD45.1-expressing mouse are
signal-to-noise ratio.
used as target cells for CD45.2 mice. (Note: Any other congenic
marker could also be used). We here use four different CFSE con-
centrations as well as four different CTV concentrations to label
11.5.3 Calculation of the division index 16 different populations in total. Therefore, the CD45.1
+ spleno-
cytes are equally divided into four aliquots. They are than labeled
FlowJo includes a function to calculate the division index. There- with the four different CFSE concentrations. Next, each of them
fore, the software identifies the individual peaks, which are used is again divided in four aliquots, which is than labeled with a dif-
for its calculation, automatically. To our experience, the gates set ferent CTV concentration. Afterwards, 4×4 = 16 populations are
by FlowJo do not always represent the correct populations. If you ready for peptide loading. Populations without peptide loading
should decide to use this FlowJo feature, it is essential to check or which are loaded with an irrelevant peptide serve as internal
whether FlowJo set the gates correctly. If not, calculation of the standard and injection control. After loading, the cell populations
division index using the formula described above might be the will be mixed in equal numbers and injected into the immunized
better choice. mice to test their immune reaction leading to the lysis of the
injected target cells. After analysis of re-isolated splenocytes by
flow cytometry (identifying the transferred cells by their CD45.1
expression), the efficacy of cell lysis is calculated by comparing
11.6 Top tricks the numbers of cells in a certain peptide-loaded population with
the unloaded fraction (or the fraction loaded with an irrelevant
The protocol provided here is based on the use of OT-I mice and peptide).
MACS preparation of OT-I T cells. Alternatively, T cells can be iso-
lated from OT-I/Rag2−/− mice. Since these cells lack endogenous
B cells and CD4+ T cells, the proportion of CD8+ T cells is signifi- 12.2 Materials
cantly higher and to our experience, MACS purification might be
omitted. 12.2.1 Reagents
Alternatively, when no OTI cells are available, the B3Z cell
line can be used instead. After their activation, these OVA-specific Necessary reagents are listed in Table 47.
hybridoma cells also secrete IL-2, which can be measured in the
supernatants as described above. In addition, these cells express
β-gal under control of the IL-2 promoter. Hence, their activation 12.2.2 Equipment
can also be measured colorimetrically after the addition of the
chromogenic substrate CPRG. Necessary equipment is listed in Tables 48 and 49.
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Table 47. Reagents, chemicals and solutions 12.3 Step-by-step sample preparation
Reagent Manufacturer Ordering
Number 12.3.1 Preparation of stocks and solutions
Chemicals & Solutions For a list of reagents see Table 47.
Hank´s Balanced Salt Sigma H6648
Solution without calcium
and magnesium DAPI
Dulbecco´s Phosphate Sigma D8537 Dissolve DAPI 4´,6-diamidino-2´-phenylindole dihydrochloride
Buffered Saline without (DAPI) in ultrapure water to create a 1 mg/ml stock solution.
calcium and magnesium Store the solution protected from light at 4°C. Dilute the stock
RPMI-1640 Sigma R8758 solution 1:10,000 in FACS buffer (PBS+2% FCS) to create a work-
Fetal Bovine Serum (FCS) Sigma F7524 ing solution for DAPI staining of cells directly before acquisition
CellTraceTM CFSE – Cell Invitrogen C34554 at the flow cytometer.
Proliferation Kit
CellTraceTM Violet – Cell Invitrogen C34557 FCS
Proliferation Kit Quickly thaw FCS at 37°C in a water bath. Once completely
4´,6-diamidino-2- Thermo 62247
thawed, incubate for 15 min at 42°C in the water bath to destroy
phenylindole Scientific
(DAPI) complement activity. Directly filter the warm FCS through a sterile
Ammonium chloride (NH Cl) Carl Roth K298.1 0.22 μmmembrane (Corning #431118) into a sterile storage bot-4
Potassium hydrogen Carl Roth P748.1 tle (Corning #430518) and aliquot into 50 ml portions. Use asep-
carbonate (KHCO3) tic techniques during the whole procedure. Aliquoted FCS must
SIINFEKL peptide (OVA257-264) Invivogen vac-sin be stored at –20°C. Avoid freeze-thaw cycles.
Antibodies
Purified αFcγRIIB/III (2.4G2) BioLegend 101302 FACS buffer
Purified αFcγRIV (9E9) BioLegend 149502 Add 2% FCS (v/v) to Phosphate buffered saline solution (PBS).
αCD45.1-PE Biolegend 110708
αCD45.2-Alexa700 Biolegend 109822 RPMI+2% FCS (CFSE & CTV Labeling buffer)
Add 2% FCS (v/v) to RPMI medium.
Table 48. Necessary equipment
Equipment Company Purpose
Centrifuge ‘Allegra X-15R’ Beckman-Coulter Centrifugation of 50 ml tubes, 15 ml tubes and V-bottom
plates
Neubauer counting chamber Superior Marienfeld Cell counting
0.100 mm; 0.0025 mm2
Corning storage bottle (#430518) and Corning Sterile filtration and storage of solutions
0.22 μM sterile filter (#431118)
Sterile bench ‘Mars Safety Class 2’ Scanlaf Performance of all aseptic procedures
LSR Fortessa SORP (#647800) BD Flow cytometric analysis of single-cell suspensions
96-well V-bottom plate (651 180) Greiner bio-one Sample preparation for flow cytometry
50 ml tubes (#352070) Falcon Centrifugation of cell suspensions
15 ml tubes (#188271) Greiner bio-one Centrifugation of cell suspensions
Serological pipettes (#606180) Greiner bio-one Pipetting
FACS tube (#352008) Corning Regular FACS tubes for acquisition of single-cell suspensions
100 μm filter for 50ml tubes Greiner bio-one Isolation of single cells from tissues
(#542000)
40 μm filters for 50ml tubes Greiner bio-one Generation of single-cell suspensions from lymphoid tissues
(#542040) by passive filtration
Pestles (#309658) BD Passage of organ material via 100 μm filters
6-well plates (#140675) Thermo Scientific Storage of organs & mechanical tissue disruption
Milli-Q Advantage A10 Millipore On-demand provision of Millipore water (ddH2O)
Waterbath
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76 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 49. Detailed configuration of the used BD LSR Fortessa
Laser Filter Fluorochrome Alternatives
line
Long pass Band pass
355 nm - 379/28 BUV395 -
410LP 470/100 BUV496 DAPI, ZombieUV
690LP 740/35 BUV737 -
405 nm - 450/50 CTV V450, e450, Pacific Blue, BV421
505LP 525/50 V500 BV510, Pacific Orange
545LP 585/42 BV570 -
600LP 610/20 BV605 -
630LP 670/50 BV650 -
690LP 710/50 BV711 -
488 nm - 488/10 FSC/SSC -
505LP 530/30 CFSE FITC, GFP, A488
685LP 710/50 PerCP-e710 PerCP-Cy5.5, PerCP
561 nm - 586/15 PE CMRA
600LP 610/20 PE-Dazzle 594 PE-CF594
635LP 670/30 PE-Cy5 PE-Dye649
750LP 780/60 PE-Cy7 PE-Fire750
640 nm - 670/14 A647 APC
690LP 730/45 A700 -
750LP 780/60 APC-Cy7 APC-Fire750, APC-H7, APC-e780
CFSE stock solution αDEC205-OVA in combination with 25 μg poly(I:C) and 25 μg
Solubilize one CFSE vial (90 nmol) in 9.5 μl water-free DMSO αCD40 antibody seven to eight days before the killing assay
(final concentration 9.5 mM) and store small aliquots (e.g., 1 μl) is a suitable immunization protocol. The here exemplary used
at –20°C. Avoid freeze-thaw-cycles. antigen targeting approach allows for the specific delivery of
antigens (in this case the model antigen ovalbumin, OVA) to
CTV stock solution cDC1 DC leading to a strong cytotoxic CD8+ T cell response. For
Solubilize one CTV vial (100 nmol) in 20 μl water-free DMSO more information see also Lehmann et al. and Dudziak et al [2,
(final concentration 5 mM) and store small aliquots (e.g., 1 μl) at 3, 187]. Therefore, we inject 100 ng αDEC205-OVA, 300 ng of
–20°C. Avoid freeze-thaw-cycles. αDEC205-OVA, 100 ng Isotype-OVA, or 300 ng Isotype-OVA in
combination with 25 μg poly(I:C) and 25 μg αCD40 i. p. into
Ammonium-Chloride-Potassium (ACK) lysis buffer for ery- C57Bl/6J mice.
throcyte lysis
Dissolve NH4Cl and KHCO3 to reach final concentrations of
155 mM and 10 mM, respectively, in double distilled H2O. Adjust
the pH to 7.3. Autoclave the solution. Store at room tempera- 12.3.3 Preparation of splenocytes
ture. After opening, do not use ACK lysis buffer longer than six
weeks, since airborne CO2 in combination with carbonate lead to Within the article “Guidelines for DC preparation and flow
pH change of the buffer. Alternatively, cell-culture grade ACK lysis cytometry analysis of mouse lymphohematopoietic tissues”
buffer is also commercially available. [9] the harvesting of splenic material and the preparation of
single-cell suspensions was described in detail in the section 1.1
Generation of a SIINFEKL peptide (OVA257-264) stock solution Preparation of mouse single-cell suspensions.
Reconstitute lyophilized SIINFEKL peptide with sterile, In brief, following harvesting of spleen, tissues are transferred
endotoxin-free water to a stock concentration of 5mM under within a six well plate to a sterile working bench. All subsequent
a cell culture hood. Store aliquots at –20°C. Avoid freeze-thaw steps are performed under sterile conditions, using sterile buffers
cycles. and equipment and aseptic techniques. Following mechanical tis-
sue disruption via mincing with tweezers, cell suspensions are fil-
tered via a 100μm cell strainer without any previous digestion
12.3.2 Immunization of mice procedure. Following centrifugation and erythrocyte lysis utiliz-
ing ACK lysis buffer, cell suspensions are passively filtered via a
Mice can be immunized with your preferred regimen priming 40 μm strainer and washed three times. The resulting cell sus-
cytotoxic CD8+ T cells. E.g., antibody targeting applying 100 ng pension is ready for the labeling process.
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
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Eur. J. Immunol. 2023;53:2249925 In vivo analysis of killing efficacy by CD8+ T cell responses 77 of 96
Table 50. CFSE labeling scheme Table 51. CTV labeling scheme
Final CFSE Volume of 90 μM CFSE solution to add to Final CTV Volume of 150 μM CTV solution to add to
concentration 10*106 cells in 200 μl medium concentration 100 μl medium
15 μM 40 μl 7.5 μM 5.1 μl
3 μM 7.9 μl 1.5 μM 1 μl
0.6 μM 1.33 μl 0.5 μM 0.34 μl
0 μM 0 μl 0 μM 0 μl
the same concentration of CTV resulting in 16 differentially
12.3.4 CFSE and CTV labeling of target cells
labeled cell aliquots.
15. Dilute 1 μl of the CTV stock solution (5 mM) with 32.3 μl of
To identify the different cell populations after transfer into the
PBS to reach a concentration of 150 μM.
immunized mice, CD45.1 donor cells are labeled with different
16. Centrifuge the prepared single-cell suspension aliquots (e.g.,
concentrations of CTV and CFSE. First, splenic single-cell sus-
500–700 × g, 5–10 min, 4°C) and remove the supernatant
pensions from a B6.SJL-Ptprca Pepcb/BoyJ (CD45.1 mice on a
completely.
C57Bl/6 background).
17. Resuspend the cell pellets in 100 μl prewarmed
RMPI1640+2% FCS per aliquot. For a higher washing
1. Prewarm RMPI1640+2%FCS to 37°C. efficiency, we recommend using 15 ml tubes.
2. Divide the single-cell suspension in four aliquots containing 18. Add the respective amount of the PBS-diluted 150 μM CTV
the same number of cells (e.g., 10*106 cells). solution according to Table 51. It is important to mix imme-
3. Dilute 1 μl of the CFSE stock solution (9.5 mM) with 104.6 diately, to add the solution in the tubes one by one, and to
μl of PBS to reach a concentration of 90 μM. start with the highest CTV concentration.
4. Centrifuge the prepared single-cell suspension aliquots (e.g., 19. Incubate for 15 min at 37°C in the waterbath (including the
500–700 × g, 5–10 min, 4°C) and remove the supernatant 0 μM CTV aliquot).
completely. 20. Fill up with cold RPMI1640+2% FCS to at least 10 ml.
5. Resuspend the cell pellets in 200 μl prewarmed 21. Centrifuge the labeled cells (e.g., 500–700 × g, 5–10 min,
RMPI1640+2% FCS per 10×106 cells. For a higher washing 4°C) and remove the supernatant completely.
efficiency, we recommend using 15 ml tubes. 22. Resuspend the cells in 500 μl prewarmed RPMI1640+2%
6. Add the respective amount of the PBS-diluted 90 μM CFSE FCS.
solution according to Table 50. It is important to mix imme- 23. Incubate for 10 min at 37°C in the waterbath (including the
diately, to add the solution in the tubes one by one, and to 0 μM CTV aliquot).
start with the highest CFSE concentration. All provided vol- 24. Centrifuge the labeled cells (e.g., 500–700 × g, 5–10 min,
umes of RPMI+2% FCS and PBS diluted CFSE solution can 4°C) and remove the supernatant completely.
be scaled up and down according to the cell count. 25. Resuspend the cells in each 100 μl of prewarmed
7. Incubate for 15 min @37°C in the waterbath (including the RPMI1640+2 %FCS.
0 μM CFSE aliquot).
8. Fill up with cold RPMI1640+2% FCS to at least 10 ml and
mix. 12.3.5 Peptide loading of target cells and injection into the
Centrifuge the labeled cells (e.g., 500–700 × g, 5–10 min, immunized mice
4°C) and remove the supernatant completely.
9. Resuspend the cells in 500 μl prewarmed RPMI1640+ To investigate the killing of the target cells, the differentially
2 %FCS. labeled cell populations are pulsed with the MHC-I/CD8 peptides
10. Incubate for 10 min at 37°C in the waterbath (including the of interest. Here, we will describe an example for different con-
0 μM CFSE aliquot). centrations of SIINFEKL peptide (the dominant CD8 epitope of
11. Centrifuge the labeled cells (e.g., 500–700 × g, 5–10 min, Ovalbumin, OVA257-264), but the populations can also be loaded
4°C) and remove the supernatant completely. with different peptides and/or different concentrations.
12. Resuspend the cells in 420 μl prewarmed RPMI1640+
2 %FCS. 1. Dilute the SIINFEKL stock solution (5 mM) to 1,250 nM
13. Now, divide each of the differentially CFSE-labeled popula- (1:4,000) with prewarmed RPMI1640+2%FCS.
tions in four 100 μl aliquots (resulting in 16 different tubes). 2. Prepare an additional 12.5 nM SIINFEKL dilution (1:100
14. In the next steps, these populations will be labeled with from the 1,250 nM concentration) with prewarmed
four different CTV concentrations (0, 0.5, 1.5, and 7.5 μM). RPMI1640+2% FCS.
Therefore, each four tubes (from each CFSE labeling, namely 3. As example, we load the differentially labeled target cell pop-
15 μM, 3 μM, 0.6 μM, and 0 μM) will be labeled with ulations with four different SIINFEKL concentrations, namely
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Table 52. Overview of SIINFEKL peptide dose assignments to differentially CTV and CFSE labeled populations
0 μM CFSE 0.6 μM CFSE 3 μM CFSE 15 μM CFSE
0 μM CTV 625 nM SIINFEKL 625 nM SIINFEKL unloaded 625 nM SIINFEKL
0.5 μM CTV 160 nM SIINFEKL 160 nM SIINFEKL 2.5 nM SIINFEKL 160 nM SIINFEKL
1.5 μM CTV 40 nM SIINFEKL 40 nM SIINFEKL 40 nM SIINFEKL 40 nM SIINFEKL
7.5 μM CTV 2.5 nM SIINFEKL 2.5 nM SIINFEKL 160 nM SIINFEKL 2.5 nM SIINFEKL
625 nM, 160 nM, 40 nM, and 2.4 nM. Additionally, we pre- 18. Inject the mixed target cell populations intravenously into
pare unloaded target cells (0 nM) as a reference. To minimize immunized mice.
the effect of the CFSE/CTV labeling on the killing efficiency,
we equally distribute the SIINFEKL concentrations to higher
and lower labeled target cell populations and use replicates. 12.3.6 Analysis of killing efficacy
Please see Table 52 for clarification.
4. Add to 100 μl of the respective target cell population the To investigate the killing of the target cells, splenic single-cell
following amounts (Table 53) of the SIINFEKL dilutions and suspensions will be generated and analyzed for their content of
mix immediately: injected target cell populations. Splenic single-cell suspensions
5. Incubate for 30 min at 37°C in the water bath. will be generated as described within article Guidelines for DC
6. Fill up with RPMI1640+2% FCS to at least 10 ml and mix. preparation and flow cytometry analysis of mouse lympho-
7. Centrifuge (e.g., 500–700 × g, 5–10 min, 4°C) and remove hematopoietic tissues [9] in section 1.1 Preparation of mouse
the supernatant completely. single-cell suspensions and as summarized under point 12.3.3
8. Resuspend the cells in 1,000 μl RPMI1640+2% FCS, fill up within this section. For effective killing, spleens of mice are har-
to at least 10 ml with RPMI1640+2% FCS and mix. vested 8 to 16 h after target cell transfer.
9. Centrifuge (e.g., 500–700 × g, 5–10 min, 4°C) and remove In brief, following harvesting of spleen, tissues are transferred
the supernatant completely. into a 6-well plate. Subsequent to mechanical tissue disruption via
10. Resuspend the cells in 1,000 μl PBS and take an aliquot for mincing with tweezers, cell suspensions are filtered via a 100 μm
cell counting before filling up to at least 10 ml with PBS. cell strainer without any previous digestion procedure. Follow-
11. Determine the number of cells in each target cell population. ing centrifugation and erythrocyte lysis utilizing ACK lysis buffer,
12. Centrifuge (e.g., 500–700 × g, 5–10 min, 4°C) and remove cell suspensions are passively filtered via a 40 μm strainer and
the supernatant completely. washed three times. The resulting cell suspension is ready for the
13. Resuspend the cells in a small volume (e.g., 100 μl) to mix labeling process.
all target cell populations in equal numbers (1:1:1:1:1:1 …
1:1:1:1:1 ratio). 1. Following the preparation of single-cell suspensions, transfer
14. Fill up to at least 10 ml with PBS. 10×106 cells per spleen to a 96-well V-bottom plate;
15. Centrifuge (e.g., 500–700 × g, 5–10 min, 4°C) and remove 2. Before centrifugation of the samples, prepare the Fc receptor-
the supernatant completely. blocking solution containing purified αFcγRIIB/III (clone
16. Resuspend the cells in room temperature (20°C) PBS for the 2.4G2; stock 0.5 mg/ml) and αFcγRIV (clone 9E9; stock
injection into the immunized mice (about 2–10*106 cell in 0.5 mg/ml) in a dilution of 1:400;
50–100 μl per mouse). 3. Centrifuge the sample containing 96-well V-bottom plates at
17. Check a small aliquot of the cells at your flow cytometer to 700 × g for 5 min at 4°C. All following centrifugations are
ensure their quality, labeling, and viability. carried out using these settings;
4. Resuspend cells in 50 μl of the Fc receptor-blocking solution;
5. Incubate the samples for 5 min at 4°C.
Table 53. Overview of added peptide amounts for peptide pulsing
6. In the meantime, prepare the antibody staining cocktail.
Final SIINFEKL To add Therefore, use 50 μl of FACS buffer per sample and add
concentration the antibodies listed in Table 54 in the indicated dilution;
Please note that antibody dilutions are dependent on the
625 nM SIINFEKL 100 μl of the 1,250 nM dilution
160 nM SIINFEKL 74.4 μl prewarmed RPMI160+2% FCS +
Table 54. Antibody staining mix for flow cytometry
25.6 μl of the 1,250 nM dilution
40 nM SIINFEKL 93.6 μl prewarmed RPMI160+2% FCS + Fluorophore/ Antigen Clone #Catalog Company Dilution
6.4 μl of the 1,250 nM dilution Labeling
2.5 nM SIINFEKL 60 μl prewarmed RPMI160+2% FCS + 40
μl of the 12.5 nM dilution PE CD45.1 A20 110708 Biolegend 1:1,000
unloaded 100 μl prewarmed RPMI1640+2% FCS Alexa700 CD45.2 104 109822 Biolegend 1:100
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Figure 29. Exemplary gating strategy for the transferred cells in the immunized mice. 2×106 target cells we transferred intravenously into immu-
nized C57Bl/6J mice. 16 h later, splenic single-cell suspensions were prepared and 10×106 splenocyte were stained with CD45.1-PE (1:1,000) and
CD45.2-Alexa700 (1:100). After washing, cells were analyzed using a BD LSR Fortessa SORP flow cytometer. First, doublet cells (ration SSCA/FSCW),
dead cells (DAPI positive) as well as debris were excluded. Next, transferred cells were identified as CD45.1+ and CD45.2−. From these cells, the
16 differentially labeled transferred target cell populations were identified according to their CFSE and CTV fluorescence. Here, we present one
exemplary gating strategy after transfer of the CD45.1 target cell population mixture into a C57BI/6J mouse. The data was acquired on a BD LSR
Fortessa SORP flow cytometer and evaluated with FlowJo v10.8 software.
flow cytometer and its setup; Suggested antibody dilutions 12.5 Pitfalls
were optimized for a BD LSR Fortessa equipped with 355 nm,
405 nm, 488 nm, 561 nm, and 640 nm laser lines; Problem: The labeling of the target cell population is not uni-
7. Centrifuge the sample containing 96-well V-bottom plates at form enough leading to difficulties in the separation after
700 × g for 5 min at 4°C; transfer.
8. Discard the supernatant, keep the plate up-side down and dip Potential solution:
on a paper towel; Ensure the direct mixing after adding the CFSE or CTV dye to the
9. Resuspend each sample in 50 μl of primary antibody staining cells.
mix and incubate for 30 min at 4°C; Adapt the PMT voltages to maximize the separation of the dif-
10. Fill up with 100 μl FACS buffer per well and centrifuge; ferent populations.
11. Discard the supernatant, keep the plate up-side down and dip Re-titrate the concentration of CTV and/or CFSE to optimize it
on a paper towel; for you flow cytometer.
12. Resuspend each sample in 160 μl FACS buffer;
13. Centrifuge at 700 × g and 4°C for 5 min;
14. Discard the supernatant, keep the plate up-side down and dip
on a paper towel;
15. Resuspend each sample in 160 μl FACS buffer;
16. Centrifuge at 700 × g and 4°C for 5 min;
17. Resuspend each sample in 100 μl of FACS buffer;
18. Samples are ready for acquisition at a flow cytometer;
19. Add 180 μl of FACS buffer containing DAPI in a dilution of
1:10,000 directly before acquisition;
20. After performing cytometer setup, acquire data at an appro-
priate flow cytometer;
12.4 Data analysis
Data of 2.5×106 cells was acquired utilizing a BD LSR Fortessa
SORP flow cytometer (configuration see Table 49) and analyzed Figure 30. Exemplary data for lysis of target cells after immunization.
by the commercial software FlowJo v10.8 (BD). After exclusion Eight weeks old, female C57Bl/6J mice were immunized with either 100
of doublets, debry, and dead cells, transferred cells were iden- ng or 300 ng of the targeting antibody αDEC205-OVA or the respectiveIsotype control (Isotype-OVA) in combination with 25 μg poly(I:C) and
tified as CD45.1+ CD45.2− (see Fig. 29). These cells were ana- 25 αCD40 antibody intraperitonally. Threeweeks later, 2×106 target cells
lyzed for their CFSE and CTV fluorescence and the number of we transferred intravenously into immunized C57Bl/6J mice. Another
6
cells within the 25 different clusters were extracted. The number 16 h later, splenic single-cell suspensions were prepared and 10×10splenocyte were stained with CD45.1-PE (1:1,000) and CD45.2-Alexa700
of not peptide loaded cells served as control for injection efficiency (1:100) and analyzed as described in Fig. 29. The cell numbers in each
as well as unspecific killing. An exemplary gating is displayed in transferred population were evaluated with FlowJo v10.8 software and
Fig. 29. Lysis for the different SIINFEKL concentrations is calcu- the lysis of the target cells was calculated using a classical Excel sheetby dividing the number of SIINFEKL labeled cells by the number of unla-
lated as percentage of non-peptide-loaded cells (example data are beled cells re-isolated from each mouse. Presented is the median spe-
presented in Fig. 30). cific depletion of the target cells of
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80 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Problem: No killing of target cells. We here describe a protocol to perform mixed leukocyte
Potential solution: responses (MLR) with cell-sorted human primary blood DC and
Ensure that the target cells have the right haplotype for the used naïve CD4+ as well as CD8+ T cells by negative enrichment. DC
peptides. E.g., SIINFEKL is dependent on the H-2Kb molecule. can be stimulated with TLR ligands prior to the co-culture in order
Therefore, the target cells as well as the immunized mice need to to analyse the effect of TLR-induced activation on the capacity of
express H-2Kb alloantigen. To ensure labeling of the target cells the DC to induce proliferation as well as polarization of the T cells.
with SIINFEKL, an αSIINFEKL bound to H-2Kb antibody (clone Here, the TLR ligand R848 was used as it is able to induce IL-
25-D1.16, available from Biolegend, e.g., 141606 for an APC con- 12 secretion by human DC subpopulations, which should induce
jugate), might be used. Th1 polarization as well as IFNγ secretion by CD4+ and CD8+ T
cells, respectively. However, also ligands for other pattern recog-
nition receptors might be used, which can influence the secretion
12.6 Top tricks of cytokines and, thereby, the phenotype of the induced T cell
response [188].
In this protocol, we use two different dyes to label 16 to 25 dif-
ferent populations. To enhance the value of the assay, a third fluo-
rescence dye can be added (leading to the distinction of 64 to 125 13.2 Materials
different target populations).
In the example data provided here, we only used one well- 13.2.1 Reagents
defined peptide, namely SIINFEKL, from Ovalbumin in different
concentrations. This protocol can be easily adapted to: 1) loading
A complete list of reagents is provided in Table 55. Further,
of different peptides, 2) more peptide concentrations (to asses
detailed protocols for cell-sorted primary DC as well as PBMCs
avidity), or even to peptide libraries.
of a HLA-mismatched blood donor are detailed in [156]
Furthermore, this assay can be applied to evaluate memory-
sections 3 “Cell sorting of primary human DC” and section
recall-responses or responses in different organs, such as lung,
1.1 “Isolation of mononuclear cells from peripheral blood”,
liver, etc. If assessing memory CD8+ T cell-mediated killing
respectively.
responses, we suggest to evaluate the optimal time point after
target cell transfer as a memory re-call might need longer than
the usual 8 to 16 hours.
13.2.2 Equipment
The assay can also be extended to assess NKT cell-mediated
killing, e.g., loading target cells with lipids of interest, such as
Necessary equipment is listed in Tables 56 and 57.
αGalCer.
As the methods focus solely on the number of killed cells, it can
be extended to tumor cell killing and NK-mediated killing by using
another target cell mixture. We recommend to always add spleen 13.3 Step-by-step sample preparation
cells as one population as the can serve as optimal control for
injection efficiency (as no-target cell population) and unspecific 13.3.1 Preparation of stocks and solutions
loss.
DC/T cell medium
Add 10% human sera type AB (v/v), 1% non-essential amino
13 Measuring naïve T cell responses acids, 1% Penicillin-Streptomycin (v/v), 1% L-Glutamine (v/v),
induced by human primary DC and 1% sodium pyruvate (v/v) to 500 ml RPMI-1640.
13.1 Introduction R848
Prepare a 1 mg/ml stock solution of R848 by adding 500 μl of
Dendritic cells (DC) are the main regulators of immunity due to endotoxin-free water to 500 μg of R848. Vortex the solution until
their capacity to induce naïve T cell responses [188, 189]. In it is completely dissolved. Prepare aliquots and store them at –
mice, DC subpopulations show clear differences in their capacity 20°C.
to polarize naïve CD4+ T cells as well as to cross-present antigens
to CD8+ T cells in vivo [2, 190, 191]. Here, cDC1 mainly induce EasySep Buffer
Th1 CD4+ T cell responses and are the main cross-presenting DC Add 2% human sera type AB and 1 mM EDTA to 500 ml PBS.
subpopulations, while cDC2 preferentially induce Th2 as well as
Th17 CD4+ T cell responses [2, 190–193]. While all human DC CFSE
subpopulations seem to be able to cross-present antigens in vitro, To prepare a 10 mM stock solution of CFSE, add 9 μl of DMSO to
results on polarization of CD4+ T cells are quite heterogeneous the CellTraceTM CFSE dye and mix well. Make 1 μl aliquots and
[194–199]. store them at –20°C.
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Table 55. List of needed reagents for the preparation of single cells from human lymphoid tissues
Reagent Manufacturer Ordering number
Antibodies
αCD3 BUV395 (UCHT1) BD Biosciences 563548
αCD4 BV510 (OKT4) BioLegend 317444
αCD8 BUV737 (SK1) BD Biosciences 564629
αCD25 PerCP/Cy5.5 (M-A251) BioLegend 356112
αIL-2 PE/Dazzle594 (MQ1-17H12) BioLegend 500344
αIL-4 A647 (8D4-8) BioLegend 500712
αIL-17A BV605 (BL168) BioLegend 512326
αIL-10 PE (JES3-9D7) BioLegend 501403
αIFNγ BV421 (4S.B3) BioLegend 502532
αTNFα PE/Cy7 (MAb11) BioLegend 502930
αGranzyme B A647 (GB11) BioLegend 515406
αPerforin PE (B-D48) BioLegend 353304
Mouse IgG1 isotype control PerCP/Cy5.5 (MOPC-21) BioLegend 400150
Rat IgG2a isotype control PE/Dazzle594 (RTK2758) BioLegend 400558
Mouse IgG1 isotype control A647 (MOPC-21) BioLegend 400136
Mouse IgG1 isotype control BV605 (MOPC-21) BioLegend 400162
Rat IgG1 isotype control PE (G0114F7) BioLegend 401906
Mouse IgG1 isotype control BV421 (MOPC-21) BioLegend 400158
Mouse IgG1 Isotype control PE/Cy7 (MOPC-21) BioLegend 400126
Mouse IgG1 isotype control PE (MOPC-21) BioLegend 400140
Chemicals & Solutions
Dulbecco´s Phosphate Buffered Saline without calcium and magnesium Sigma-Aldrich D8537
RPMI-1640 Medium Sigma-Aldrich R8758
Human Serum type AB (male) Sigma-Aldrich H4522
UltraPureTM 0.5M EDTA, pH 8.0 Thermo Fisher Scientific 15575020
Trypan blue Sigma-Aldrich T6146
R848 (Resiquimod) Invivogen tlrl-r848
4´,6-diamidino-2-phenylindole (DAPI) Thermo Scientific 62247
MEM Non-Essential Amino Acids Solution (100X) Thermo Fisher Scientific 11140050
Penicillin-Streptomycin Sigma-Aldrich P4333
L-Glutamine Sigma-Aldrich G7513
Sodium Pyruvate Sigma-Aldrich S8636
Phorbol myristate acetate (PMA) Invivogen tlrl-pma
Ionomycin Invivogen inh-ion
Brefeldin A Solution (1,000X) BioLegend 420601
Kits
EasySepTM Human Naïve CD4+ T Cell Isolation Kit II Stemcell Technologies 17555
EasySepTM Human Naïve CD8+ T Cell Isolation Kit II Stemcell Technologies 17968
CellTraceTM CFSE Cell Proliferation Kit, for flow cytometry Thermo Fisher Scientific C34554
DynabeadsTM Human T-Activator CD3/CD28 for T Cell Expansion and Thermo Fisher Scientific 11131D
Activation
Zombie UVTM Fixable Viability Kit BioLegend 423108
Cytofix/CytopermTM Fixation/Permeabilization Kit BD Biosciences 554714
Phorbol myristate acetate (PMA) Restimulation medium
Prepare a 5 mg/ml stock solution of PMA by adding 1 ml of DMSO Add 1 μg/ml PMA, 5 μg/ml Ionomycin as well as 1% Brefeldin A
to 5 mg of PMA. Vortex until PMA is completely dissolved. Prepare solution (1000x) to DC/T cell medium.
aliquots and store them at –20°C.
Zombie UVTM Fixable Viability Dye
Ionomycin Add 100 μl of DMSO to one vial of Zombie UVTM Dye and pipet up
Prepare a 10 mg/ml stock solution of Ionomycin by adding 100 μl and down until it is completely dissolved. Prepare 10 μl aliquots
of DMSO to 1 mg of Ionomycin. Vortex until Ionomycin is com- and store at –80°C.
pletely dissolved. Prepare aliquots and store them at –20°C.
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82 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 56. Necessary equipment
Equipment Company Purpose
Centrifuge “Allegra X-15R” Beckman-Coulter Centrifugation of 50 ml tubes, 15 ml tubes and
V-bottom plates
Incubator “HERAEUS BBD6220” Thermo Scientific Cabinet style incubator with 5% CO2 and 96%
relative humidity for the lymphoid tissue
digestion
LSR Fortessa (#647800) BD Flow cytometric analysis of single-cell
suspensions
Neubauer chamber 0.100 mm; 0.0025 mm2 Superior Marienfeld Cell counting
Sterile bench “Mars Safety Class 2” Scanlaf Performance of all aseptic procedures
96 Well Cell Culture Plate (V-bottom) (#651 180) Greiner bio-one Plates for the stimulation of sorted DC in sterile
conditions
Clear Flat-Bottom Immuno Non-sterile 96-Well Thermo Scientific Performance of the LDH assay
Plates (#442404)
50 ml tubes (#352070) Falcon Preparation of 1 x Wash buffer (LEGENDplex
assay)
14 ml tubes, round bottom, (#187262) Greiner bio-one Isolation of naïve CD4+ and CD8+ T cells
Serological pipettes (#606180) Greiner bio-one Pipetting
“The Big Easy” EasySepTM Magnet (#18001) Stemcell Isolation of naïve CD4+ and CD8+ T cells
Technologies
FACS buffer 13.3.2 Measuring naïve T cell responses using mixed
Add 2% human serum type AB (v/v) to 500 ml of Phosphate leukocyte reactions with primary DC
buffered saline solution (PBS).
13.3.2.1 Stimulation of primary DC with TLR ligands.
1× Perm/Wash Buffer
Dilute the 10× Perm/Wash Buffer (Cytofix/CytopermTM Fixa- 1. Isolate primary human cDC1, cDC2, pDC, and monocytes as
tion/Permeabilization Kit) 1:10 with ddH O and mix well by described in section 3 “Cell sorting of primary human DC” in2
inverting. [156]. Resuspend the isolated DC in DC medium to a final
concentration of 2×105 cells/ml.
Table 57. Detailed configuration of the used BD LSR Fortessa
Laser line Filter Fluorochrome Alternatives
Long pass Band pass
355 nm - 379/28 BUV395 -
410LP 470/100 DAPI BUV496, ZombieUV
690LP 740/35 BUV737 -
405 nm - 450/50 BV421 V450, e450, Pacific Blue, CTV
505LP 525/50 BV510 V500, Pacific Orange
545LP 585/42 BV570 -
600LP 610/20 BV605 -
630LP 670/50 BV650 -
690LP 710/50 BV711 -
488 nm - 488/10 FSC/SSC -
505LP 530/30 FITC A488, GFP, CFSE
685LP 710/50 PerCP-Cy5.5 PerCP-e710, PerCP
561 nm - 586/15 PE Cy3, CMRA
600LP 610/20 PE-Dazzle 594 PE-CF594
635LP 670/30 PE-Cy5 PE-Dye649
750LP 780/60 PE-Cy7 PE-Fire750
640 nm - 670/14 APC A647
690LP 730/45 A700 -
750LP 780/60 APC-Fire750 APC-Cy7, APC-H7, APC-e780
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Eur. J. Immunol. 2023;53:2249925 Measuring naïve T cell responses induced by human primary DC 83 of 96
2. Seed 10,000 (50 μl) primary cDC1, cDC2, pDC, or monocytes 19. Leave the tube inside the magnet, while pouring off the cell
into the wells of 96-well plate (U-bottom). suspension into a new 14 ml tube.
3. Prepare a 10 μg/ml solution of R848 by diluting R848 stock 20. Remove the tube from the magnet and throw it away. Place
solution 1:100 in DC/T cell medium. the new 14 ml tube (step 19) without lid into the magnet and
4. Add 50 μl of diluted R848 (final concentration of 5 μg/ml) incubate again for 3 min at RT.
or 50 μl of DC/T cell medium to the DC or monocytes in the 21. Leave the tube inside the magnet, while pouring off the cell
96-well plate. suspension into a new 14 ml tube.
5. Incubate for 3 h at 37°C in the incubator to induce activation 22. The new 14 ml tube (Step 21) contains the isolated naïve
or not in the cultured DC. CD8+ T cells. Count the cells using a Neubauer chamber.
6. During incubation, proceed with isolation of naïve CD4+ T
cells (13.3.2.2.1.) or naïve CD8+ T cells (13.3.2.2.2.).
13.3.2.3 Labeling of isolated naïve T cells with CFSE.
13.3.2.2 Isolation of naïve T cells. 23. Add 199 μl of PBS to an aliquot of the 10 mM CFSE stock
solution (dilutes the stock to a concentration of 50 μM).
24. Resuspend 5×106 naïve CD4+ (13.2.2.1.) or CD8+
13.3.2.2.1 Isolation of Naïve CD4+ T cells. (13.2.2.2.) T cells in 90 μl of pre-warmed RPMI-1640 after
washing the cells with PBS twice. Add 10 μl (1:10 dilution;
7. Transfer 2×108 PBMCs of a HLA-mismatched donor into final concentration of 5 μM) of the diluted CFSE (step 23) to
14 ml round bottom tubes. Add EasySep buffer to adjust to a the T cells. Mix well and incubate at 37°C for 20 min.
final concentration of 5×107 cells/ml. 25. Fill up to 10 ml with ice-cold DC/T cell medium to stop label-
8. Add 200 μl (50 μl/ml cell suspension) EasySepTM Human ing of the T cells. Centrifuge for 5 min with 520 × g at 4°C.
Naïve CD4+ T Cell Isolation Cocktail II (Component 26. Wash the cells with 10 ml of DC/T cell medium and count the
#17555C) to the cell suspension, mix well and incubate for cells using a Neubauer chamber. Adjust the cell concentration
5 min at RT. to 5×105 cells/ml in DC/T cell medium.
9. Vortex the EasySepTM Dextran RapidSpheresTM (Component
#50103) for at least 30 s. Then, add 200 μl (50 μl/ml cell
suspension) of the EasySepTM Dextran RapidSpheresTM to the 13.3.2.4 Performing mixed leukocyte reactions.
cell suspension and fill up to 10 ml using EasySep buffer.
10. Remove the lid of the tube and place it into “The Big Easy” 27. Pre-warm DC/T cell medium as well as the T cells to 37°C.
EasySepTM Magnet and incubate it at RT for 3 min. 28. Remove the 96-well plate after the 3 h incubation time from
11. Leave the tube inside the magnet, while pouring off the cell the incubator (step 5). Centrifuge for 5 min with 520 × g
suspension into a new 14 ml tube. at RT. Carefully remove the medium from each well using a
12. Remove the tube from the magnet and throw it away. Place pipette.
the new 14 ml tube (step 11) without lid into the magnet and 29. Resuspend the DC in 100 μl of pre-warmed DC/T cell
incubate again for 3 min at RT. medium and centrifuge for 5 min with 520 × g at RT. Care-
13. Leave the tube inside the magnet, while pouring off the cell fully remove the medium from each well using a pipette.
suspension into a new 14 ml tube. 30. If you want to analyze naïve CD4
+ T cell responses, add
14. The new 14 ml tube (Step 13) contains the isolated naïve 200 μl (100,000 cells) of the pre-warmed CFSE-labeled naïve+
CD4+ T cells. Count the cells using a Neubauer chamber. CD4 T cells to each well. If you want to analyze naïve
CD8+ T cell responses, add 200 μl (100,000 cells) of the pre-
warmed CFSE-labeled naïve CD8+ T cells to each well.
13.3.2.2.2 Isolation of naïve CD8+ T cells. 31. Additionally, add 200 μl of the T cells to six wells of the plate.
Add 2.5 μl αCD3/CD28-coated Dynabeads (corresponding to
15. Transfer 2×108 PBMCs of a HLA-mismatched donor into 100,000 beads) to three of the wells as positive control, while
14 ml round bottom tubes. Add EasySep buffer to adjust to a the other three wells serve as negative control.
final concentration of 5×107 cells/ml. 32. Co-culture the DC and T cells (corresponds to a ratio of 1:10)
16. Add 200 μl (50 μl/ml cell suspension) of EasySep for 6 days at 37°C in the incubator (dependent on the aim of
Human Naïve CD8+ T Cell Isolation Cocktail (Component the analysis, also shorter periods of co-culture are possible;
#19258C), mix well, and incubate for 5 min at RT. see 13.6 Top tricks). During the culture, regularly check the
17. Vortex the EasySepTM Dextran RapidSpheresTM (Component plate for consumed media (color change to orange-yellow).
#50103) for at least 30 s. Then, add 200 μl (50 μl/ml cell If the medium is consumed, centrifuge the plate for 5 min
suspension) of the EasySepTM Dextran RapidSpheresTM to the with 300 × g at RT. Remove 100 μl of the medium from the
cell suspension and fill up to 10 ml using EasySep buffer. wells and add 100 μl fresh pre-warmed DC/T cell medium.
18. Remove the lid of the tube and place it into “The Big Easy” Gently resuspend the well and proceed the co-culture in the
EasySepTM Magnet and incubate it at RT for 3 min. incubator.
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84 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Table 58. Extracellular antibody staining panel for MLR with naïve Table 60. Intracellular antibody staining panel for MLR with naïve
CD4+ T cells CD4+ T cells
Fluorophore/ Antigen Clone #Catalog Company Dilution Fluorophore/ Antigen Clone #Catalog Company Dilution
Labeling Labeling
BUV395 CD3 UCHT1 563548 BD Bio- 1:100 PE/Dazzle594 IL-2 MQ1- 500344 BioLegend 1:200
sciences 17H12
BV510 CD4 OKT4 317444 BioLegend 1:100 A647 IL-4 8D4-8 500712 BioLegend 1:100
PerCP/Cy5.5 CD25 M-A251 356112 BioLegend 1:200 BV605 IL-17A BL168 512326 BioLegend 1:100
PE IL-10 JES3- 501403 BioLegend 1:50
9D7
Table 59. Extracellular antibody staining panel for MLR with naïve
CD8+ T cells BV421 IFNγ 4S.B3 502532 BioLegend 1:100
PE/Cy7 TNFα MAb11 502930 BioLegend 1:200
Fluorophore/ Antigen Clone #Catalog Company Dilution
Labeling Table 61. Intracellular antibody staining panel for MLR with naïve
CD8+ T cells
BUV395 CD3 UCHT1 563548 BD Bio- 1:100
sciences Fluorophore/ Antigen Clone #Catalog Company Dilution
BUV737 CD8 SK1 564629 BD Bio- 1:100 Labeling
sciences
PerCP/Cy5.5 CD25 M-A251 356112 BioLegend 1:200 PE/Dazzle594 IL-2 MQ1- 500344 BioLegend 1:200
17H12
A647 Granz- GB11 515406 BioLegend 1:100
13.3.2.5 Flow cytometric analysis of T cell proliferation, activation, yme B
and polarization. BV605 IL-17A BL168 512326 BioLegend 1:100
PE Perforin B-D48 353304 BioLegend 1:50
BV421 IFNγ 4S.B3 502532 BioLegend 1:100
33. For each well, prepare 20 μl of restimulation medium by
PE/Cy7 TNFα MAb11 502930 BioLegend 1:200
diluting PMA (1:5,000), Ionomycin (1:2,000), and Brefeldin
A (1:100) in DC/T cell medium. In case you prepare less than
2,500 μl, perform pre-dilutions of PMA and Ionomycin. Pre- 41. Remove the supernatant and resuspend all samples (with
warm the restimulation medium to 37°C. DC) in 50 μl of the prepared master mix as well as two wells
34. After the incubation time, add 20 μl of restimulation medium of the positive (with CD3/CD28 Dynabeads) and negative (T
to each well to reach a final concentration of 100 ng/ml PMA, cells only) controls. Resuspend the remaining third well of
500 ng/ml Ionomycin, and 0.1% Brefeldin A. Resuspend the the positive as well as negative control with the antibody mix
wells and incubate for 6 h at 37°C. with the mouse IgG1 isotype control PerCP/Cy5.5. Incubate
35. Transfer the content of each well to a 96-well V-bottom plate. for 15 min at 4°C.
Centrifuge the plate for 5 min with 520 × g at 4°C. Remove 42. Add 100 μl FACS buffer to each well and mix. Centrifuge the
the supernatant. plate for 5 min with 520 × g at 4°C.
36. Rinse the U-bottom plate with PBS to collect cells that 43. Remove the supernatant and resuspend the cells in 100 μl
remained in the wells and use it to resuspend the cells in FACS buffer. Centrifuge the plate for 5 min with 520 × g at
the corresponding well on the 96-well V-bottom plate. 4°C.
37. Centrifuge the plate for 5 min with 520 × g at 4°C. Remove 44. Remove the supernatant and resuspend the cells in
the supernatant and resuspend the cells in PBS. Centrifuge 80 μl Cytofix/Cytoperm Buffer (Cytofix/CytopermTM Fix-
the plate for 5 min with 520 × g at 4°C. ation/Permeabilization Kit, BD Biosciences). Incubate for
38. Remove the supernatant and resuspend the cells in 50 μl 20 min at 4°C.
PBS + Zombie UVTM Fixable Viability Dye (1:200). Incubate 45. Add 100 μl FACS buffer and centrifuge for 5 min with
on ice for 15 min in the dark. 520 × g at 4°C.
39. Add 100 μl FACS buffer and centrifuge for 5 min with 46. Remove the supernatant and resuspend the cells in
520 × g at 4°C. 1× Perm/Wash buffer. Incubate for 5 min at RT. Centrifuge
40. Remove the supernatant and resuspend the cell pellet in for 5 min with 520 × g at RT.
100 μl FACS buffer. Centrifuge for 5 min with 520 × g at 4°C. 47. Remove the supernatant and resuspend the cells in
During centrifugation, prepare a master mix (50 μl/sample) 1× Perm/Wash buffer. Incubate for 5 min at RT. Centrifuge
for staining CD4+ T cells (Table 58) or CD8+ T cells (Table for 5 min with 520 × g at RT.
59) dependent on which cells were used for the MLR. Dilute 48. During the incubation time, prepare the staining mix for
the antibodies according to Table 58 or Table 59 in FACS intracellular staining of cytokines. Dilute the antibodies
buffer. Further, prepare one sample with mouse IgG1 isotype according to Table 60 (CD4+ T cells) or Table 61 (CD8+ T
control PerCP/Cy5.5 instead of αCD25 PerCP/Cy5.5. cells) in 1× Perm/Wash buffer (50 μl/sample). Prepare an
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Table 62. Isotype control staining for MLR with naïve CD4+ T cells
Fluorophore/Labeling Isotype Clone #Catalog Company Dilution
PE/Dazzle594 Rat IgG2a RTK2758 400558 BioLegend 1:200
A647 Mouse IgG1 MOPC-21 400136 BioLegend 1:125
BV605 Mouse IgG1 MOPC-21 400162 BioLegend 1:160
PE Rat IgG1 G0114F7 401906 BioLegend 1:400
BV421 Mouse IgG1 MOPC-21 400158 BioLegend 1:100
PE/Cy7 Mouse IgG1 MOPC-21 400126 BioLegend 1:200
Table 63. Isotype control staining for MLR with naïve CD8+ T cells
Fluorophore/Labeling Isotype Clone #Catalog Company Dilution
PE/Dazzle594 Rat IgG2a RTK2758 400558 BioLegend 1:200
A647 Mouse IgG1 MOPC-21 400136 BioLegend 1:100
BV605 Mouse IgG1 MOPC-21 400162 BioLegend 1:160
PE Mouse IgG1 MOPC-21 400140 BioLegend 1:160
BV421 Mouse IgG1 MOPC-21 400158 BioLegend 1:100
PE/Cy7 Mouse IgG1 MOPC-21 400126 BioLegend 1:200
isotype control mix according to Table 62 (CD4+ T cells) gle cells using FSC-A/FSC-H. Exclude dead cells using
or Table 63 (CD8+ T cells) in 1× Perm/Wash buffer for 4 Zombie UV. Gate for CD4+ T cells using CD3 as well as
samples. CD4.
49. Resuspend the cells (DC with T cells) as well as one well 3. To identify proliferated and activated CD4+ T cells, select
(stained with master mix in extracellular staining mix) of the the gated CD4+ T cells and analyze for CFSE as well
positive (with CD3/CD28 Dynabeads) and negative (T cells as CD25. Using a quadrant gate, divide the T cells into
only) controls in 50 μl staining mix. Resuspend the remain- not proliferated/not activated (CFSE+/CD25−), not prolif-
ing two wells in 50 μl of the isotype control mix. Incubate for erated/activated (CFSE+/CD25+), proliferated/not activated
30 min at RT. (CFSE−/CD25−) and proliferated/activated (CFSE−/CD25+)
50. Add 100 μl 1× Perm/Wash buffer to each well. Incubate for as shown in Fig. 31B. Set the gate using the negative con-
5 min at RT. trol wells (T cells without Dynabeads without DC). Here, all
51. Centrifuge for 5 min with 520 × g at RT. Remove the super- T cells should be in the CFSE+/CD25− quadrant (Fig. 31B,
natant and resuspend in 100 μl Perm/Wash buffer. Incubate left panel).
for 5 min. 4. Assign the gating to the positive control well (T cells with
52. Centrifuge for 5 min with 520 × g at RT. Remove the super- 100k Dynabeads) and assure that the majority of the T cells
natant and resuspend in 100 μl Perm/Wash buffer. Incubate is now in the upper left quadrant (CFSE−/CD25+) (Fig. 31B,
for 5 min. middle panel). If this is the case, transfer the gate to all sam-
53. Centrifuge for 5 min with 520 × g at RT. Remove the super- ples.
natant and resuspend in 100 μl FACS buffer. 5. HLA-mismatched T cells cultured with DC should show prolif-
54. Centrifuge for 5 min with 520 × g at RT. Remove the super- eration in between negative and positive control. Thus, cells
natant and resuspend in 100 μl FACS buffer. should be in the lower right quadrant corresponding to not
55. Centrifuge for 5 min with 520 × g at RT. Remove the super- proliferated/not activated and cells in the upper left quadrant
natant and add 100 μl FACS buffer to each well. Acquire the corresponding to proliferated and activated T cells (see Fig.
samples on an LSRFortessa (BD Biosciences; Table 57). 31B, right panel). Using the statistics function of FlowJo,
export an Excel file with the percentage of proliferated CD4+
T cells by adding “Frequency of parent” to the CFSE−CD25+
13.4 Data analysis gate.
6. Select the proliferated and activated T cells (CFSE−/CD25+)
13.4.1 Analysis of naïve CD4+ T cell proliferation, and analyze for intracellular cytokines. To account for unspe-
activation, and polarization cific binding of the antibodies, use the positive control (T cells
with Dynabeads) stained with CD25-PerCP/Cy5.5 in the first
1. Export the files in fcs 3.1 format. Analyze the data using staining step and the isotype controls in the second staining
FlowJo (V10) or another software you prefer. step. Based on the fluorescence signal of the isotype controls,
2. For analysis, gate lymphocytes using a morphology gate set the gates either using the histogram function or using
(FSC-A/SSC-A) as shown in Fig. 31A. Then, select sin- quadrant gates in pseudocolor plots (see Fig. 31C). In the
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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86 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Figure 31. Flow cytometric analysis of CD4+ T cells after MLR with human cDC2.MLR was performed as described in 13.3.2. After restimulation of
T cells, CD4+ T cells were stained with antibodies (see Table 58). Samples were acquired using an LSRFortessa and analyzed using FlowJo (V10). (A)
After a morphology gate (FSC-A/SSC-A), doublets were excluded using (FSC-A/FSC-H). Within living cells (Zombie UV−), CD4+ T cells (CD3+CD4+)
were gated. (B) CD4+ T cells gated as in (A) were analyzed for proliferation (CFSE) and activation (CD25). Cells were divided into proliferated and
activated (CFSE−CD25+) T cells and not proliferated, not activated T cells (CFSE+CD25−) using a quadrant gate. (C-D) Proliferated and activated T
cells (CFSE−CD25+; upper left quadrant in (B)) as gated in B were analyzed for production of cytokines by intracellular flow cytometry. According to
the signal of isotype controls (C), gates were set and transferred to (D) samples stained with specific antibodies for the cytokines (see Table 60).
positive gates, less than 1% positive cells should be contained other preferred software (see Fig. 32 as an example). Sum-
for the isotype controls. marized results are shown in Table 64.
7. Apply the gating for the cytokines to all samples and add “Fre-
quency of grandparents” or “Frequency of parents” to each of
the gated cytokine+ populations. Export the frequency using 13.4.2 Analysis of naïve CD8+ T cell proliferation,
the Table function of FlowJo. activation, and phenotype
8. In Excel, correct for the signal of the isotype control by sub-
tracting the percentage of positive cells in the T cell controls
stained with isotypes from each of the samples stained with 10. Export the files in fcs 3.1 format. Analyze the data using
antibodies against the different cytokines. FlowJo (V10) or another software you prefer.
9. The results of proliferation as well as cytokine production of 11. For analysis, gate lymphocytes using a morphology gate
the T cells can now be plotted using GraphPad Prism or any (FSC-A/SSC-A) as shown in Fig. 33A. Then, select single
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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Eur. J. Immunol. 2023;53:2249925 Measuring naïve T cell responses induced by human primary DC 87 of 96
Figure 32. Measuring proliferation and polarization of CD4+ T cells after MLRwith human cDC2.MLRwas performed as described in 13.3.2. CD4+ T
cells were analyzed as shown in Fig. 31. For each population frequency of parent (Fig. 31B) or frequency of grandparent (Fig. 31C-D) was determined
and exported using FlowJo (V10). (A) Frequency of proliferated CD4+ T cells was plotted as bar graphs (+ standard deviation (SD); individual donors
shown as filled circles) using GraphPad Prism (V9). (B) Frequency of cytokine+ cells among all CD4+ T cells was plotted as bar graphs (+ standard
deviation (SD); individual donors shown as filled circles) using GraphPad Prism (V9).
cells using FSC-A/FSC-H. Exclude dead cells using Zombie In the positive gates, less than 1% positive cells should be
UV. Gate for CD8+ T cells using CD3 as well as CD8. contained for the isotype controls.
12. To identify proliferated CD8+ T cells, select the gated CD8+ 16. Apply the gating for the cytokines to all samples and add
T cells and analyze for CFSE using the histogram function. “Frequency of grandparents” or “Frequency of parents” to
Divide the T cells into not proliferated (CFSE+) and pro- each of the gated cytokine+ populations. Export the fre-
liferated (CFSE−) cells as shown in Fig. 31B. Set the gate quency using the Table function of FlowJo.
using the negative control wells (T cells without Dynabeads 17. In Excel, correct for the signal of the isotype control by sub-
without DC). Here, all T cells should be CFSE+ (Fig. 33B, tracting the percentage of positive cells in the T cell controls
left panel). stained with isotypes from each of the samples stained with
13. Assign the gating to the positive control well (T cells with antibodies against the different cytokines.
100k Dynabeads) and assure that the majority of the T cells 18. The results of proliferation as well as cytokine production
is now in the CFSE− gate (Fig. 33B, middle panel). If this is of the T cells can now be plotted using GraphPad Prism or
the case, transfer the gate to all samples. any other preferred software (see Fig. 34 as an example).
14. HLA-mismatched T cells cultured with DC should show Summarized results are shown in Table 64.
proliferation in between the negative and positive control.
Thus, some cells should be in the CFSE+ gate corresponding
to not proliferated cells, while other cells should be negative 13.5 Pitfalls
for CFSE corresponding to proliferated CD8+ T cells (see Fig.
33B, right panel). Using the statistics function of FlowJo, Problem: T cells did not proliferate after co-culture with DC
export an Excel file with the percentage of proliferated Potential solutions:
CD8+ T cells by adding “Frequency of parent” to the CFSE− 1 of 1,000-10,000 T cells recognizes foreign MHC. Thus, using
gate. 100,000 T cells for the assay should induce a clearly measurable T
15. Select the proliferated CD8+ T cells (CFSE−) and analyze cell response. In case, no proliferation (< 1% proliferated T cells)
for intracellular cytokines. To account for unspecific binding was observed, positive control wells should be analyzed. If also
of the antibodies, use the positive control (T cells with the T cells cultured with 100,000 Dynabeads did not proliferate,
Dynabeads) stained with the isotype controls in the second viability or functionality of the T cells was impaired. Thus, T cell
staining step. Based on the fluorescence signal of the isotype isolation should be repeated with fresh buffers, and T cell assay
controls, set the gates either using the histogram function performed with fresh medium. If the T cells cultured with 100,000
or using quadrant gates in pseudocolor plots (see Fig. 33C). Dynabeads proliferated (more than 80% proliferated T cells), then
Table 64. Summary of obtained results with naïve CD4+ (13.4.1) and CD8+ (13.4.2) T cells
Read out of the assays
Experimental condition Proliferation Cytokine production
CD4+ T cells TLR-stimulation of cDC2 Increased Th1 and Th2 polarization with TLR-treated cDC2
enhanced T cell proliferation
CD8+ T cells TLR-stimulation of cDC2 Increased production of IFNγ, IL-2 as well as Perforin after
enhanced T cell proliferation co-culture with TLR-treated cDC2
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Wiley-VCH GmbH
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88 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
Figure 33. Flow cytometric analysis of CD8+ T cells after MLR with human cDC2.MLR was performed as described in 13.3.2. After restimulation of
T cells, CD8+ T cells were stained with antibodies (see Table 59). Samples were acquired using an LSRFortessa and analyzed using FlowJo (V10). (A)
After a morphology gate (FSC-A/SSC-A), doublets were excluded using (FSC-A/FSC-H). Within living cells (Zombie UV−), CD8+ T cells (CD3+CD8+)
were gated. (B) CD8+ T cells gated as in (A) were analyzed for proliferation (CFSE). Cells were divided into proliferated (CFSE−) T cells and not
proliferated T cells (CFSE+) using a threshold according to the negative control (left panel). (C-D) Proliferated T cells (CFSE−) as gated in (B) were
analyzed for production of cytokines by intracellular flow cytometry. According to the signal of isotype controls (C), gates were set and transferred
to (D) samples stained with specific antibodies for the cytokines (see Table 61).
most probably T cells were cultured with autologous instead of the CFSE-staining to remove proteins, which are present in the
allogeneic DC. Repeat the assay and make sure to culture DC with buffer. Make sure that the correct number of cells was used dur-
T cells from an HLA-mismatched donor. ing CFSE-staining. If the cell concentration is too high, T cells will
only be weakly labeled with CFSE, which impairs the differentia-
Problem: Discrimination of proliferated and not proliferated tion of proliferated and not proliferated cells.
T cells is not possible
Potential solutions: Problem: High background values for isotype controls
In case discrimination of proliferated and not proliferated T cells Potential solutions:
is difficult, staining with CFSE has to be optimized. To allow If high background values for isotype controls were observed,
for discrimination, CFSE-staining of the T cells has to be high either washing of the samples was not performed accordingly
enough. Make sure that T cells were washed with PBS prior to or the concentration of the isotype control was too high. As the
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
 15214141, 2023, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/eji.202249925 by Universitätsbibliothek Mainz, Wiley Online Library on [07/03/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Eur. J. Immunol. 2023;53:2249925 Measuring naïve T cell responses induced by human primary DC 89 of 96
Figure 34. Measuring proliferation and cytokine production of CD8+ T cells after MLR with human cDC2. MLR was performed as described in
13.3.2. CD8+ T cells were analyzed as shown in Fig. 33. For each population frequency of parent (Fig. 33B) or frequency of grandparent (Fig. 33C-D)
was determined and exported using FlowJo (V10). (A) Frequency of proliferated CD8+ T cells was plotted as bar graphs (+ standard deviation (SD);
individual donors shown as filled circles) using GraphPad Prism (V9). (B) Frequency of cytokine+ cells among all CD8+ T cells was plotted as bar
graphs (+ standard deviation (SD); individual donors shown as filled circles) using GraphPad Prism (V9).
Perm/Wash buffer in the used kit contains saponin, small holes 13.7 Summary table
in the membrane are built for the intracellular staining process.
To allow unbound antibody to exit the cells, two washing steps Assay read out for co-culture of TLR-treated cDC2 and allogeneic
should be performed with Perm/Wash buffer and an incubation naive CD4+ and CD8+ T cells is summarized in Table 64
time of 5 min. Further, make sure that the concentration of the
isotype controls matches the concentration of the antibody. As
antibodies against intracellular cytokines often have concentra-
tions less than 50 μg/ml, the isotype control either has to have
the same concentration or the dilution has to be adapted. E.g. in
case the antibody has a concentration of 50 μg/ml and is used in
1:50 dilution, then an isotype control with 200 g/ml has to be Acknowledgments: Sections 1, 9, 12 and 13: The Dudziakμ
diluted 1:200. laboratory was supported by the German Research Founda-
tion [Deutsche Forschungsgemeinschaft (DFG)] (CRC1181-TPA7
261193037, DU548/5-1 420943261, TRR305-TPB5 429280966,
RTG2504 401821119, RTG2599 421758891), the Interdiszi-
13.6 Top tricks plinäres Zentrum für klinische Forschung (IZKF) (IZKF-A80,
IZKF-A87) and the Bavarian State Ministry of Science and Art
This section describes how to analyze the capacity of human DC to (Bayresq.Net-IRIS). Diana Dudziak was funded by the Agence
induce naïve T cell responses. As it becomes more and more aware Nationale de la Recherche (ANR) and the DFG (DU548/6-1
that sustained antigen stimulation can lead to T cell exhaustion 431402787).
[200], marker of T cell exhaustion (e.g. CD223 (LAG-3), CD279 Section 2: Dr. Luciana Berod received intramural funding from
(PD-1), CD366 (Tim-3)) can be added to the extracellular anti- ReALity, a joint initiative of all principal investigators of the Fac-
body staining mix (Tables 60 and 61). Further, while here an ulty of Biology, the Focus Program Translational Neurosciences
exemplary TLR ligand (R848) was used, which was shown before (FTN), the Research Center for Immunotherapy (FZI), and the
to strongly activate human primary DC [4, 153, 199, 201], also Center for Translational Vascular Biology (CTVB) at Johannes
other TLR ligands as well as tolerance-inducing ligands might Gutenberg University Mainz.
be used. In addition, blocking antibodies against select cytokines Section 4: This work was supported by the Deutsche Forschungs-
(e.g. IL-12) might be added during the co-culture of the DC and gemeinschaft (DFG), grant numbers SFB 1292-Q1 and TE599/3-1
T cells in order to determine the impact of select cytokines on to S.T., SFB 1292-TP11 and DI 2471/1-1 to U.D., SFB 1292-TP20N
proliferation and polarization of the T cells. and CL 419/2-2 to B.E.C., BA 5939/2-1 to R.A.B., as well as by the
In this protocol, DC and T cells were co-cultured for six days Research Center for Immunotherapy (FZI, Forschungszentrum für
(13.3.2.4, step 32), which leads to strong proliferation of the Immuntherapie) of the Johannes Gutenberg University Mainz.
responding T cells. In order to obtain more information about the Section 5: This work was supported by the Deutsche Forschungs-
stimulatory capacity of the different DC subpopulations, the co- gemeinschaft (DFG HI1103/1-1) to T.H.
culture of DC and T cells might be reduced to a shorter period Section 6: We thank Kasia Stefanowski for technical assistance,
(e.g., 3 days). Thereby, influences of enhanced expression of and the Core Facility Bioimaging of the Biomedical Center (BMC)
MHC-I and MHC-II molecules as well as co-stimulatory molecules of the Ludwig-Maximilian University for support. This work was
on cDC compared to pDC might be observed. However, shorter supported by the Peter Hans Hofschneider Professorship of the
period of co-culture might also affect production of cytokines as Stiftung Experimentelle Biomedizin (to J.R), by the DFG (Collab-
well as expression of exhaustion marker on the analysed T cells. orative Research Center SFB914, project A12; to J.R) and by the
© 2022 The Authors. European Journal of Immunology published by www.eji-journal.eu
Wiley-VCH GmbH
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90 of 96 Clausen BE et al. Eur. J. Immunol. 2023;53:2249925
LMU Institutional Strategy LMU-Excellent within the framework Jacobi, Lukas Heger, and Diana Dudziak; * Contributed equally;
of the German Excellence Initiative (to J.R). lead author Diana Dudziak
Section 7: This work was supported by the Human Frontier Section 13: Lukas Heger, Lukas Hatscher, Tomasz
Science Program (HFSP) RGP0032-2022, Deutsche Forschungs- Kaszubowski, and Diana Dudziak, lead author Diana Dudziak
gemeinschaft (DFG) grant number 335447717-SFB 1328
(project A20), and Forschungszentrum Medizintechnik Hamburg Data availability statement: Section 4: The mass spectrome-
(FMTHH) 04fmthh2021 to P.J.S. try proteomics data have been deposited to the ProteomeXchange
Section 8: We thank members of the Clausen laboratory for fruit- Consortium (http://proteomecentral.proteomexchange.org) via
ful discussions and critical reading of the manuscript. Work in the jPOST partner repository25 and can be accessed using
the Clausen laboratory is supported by grants from the German the dataset identifiers PXD031960 (ProteomeXchange) and
Research Foundation (Deutsche Forschungsgemeinschaft, DFG) JPST001508 (jPOST). For all other sections the data that support
to B.E.C. (CL 419/2-2 [Project Nr. 315501751], CL 419/4-1, the findings of this study are available from the corresponding
CL 419/7-1 [Project Nr. 503972215], and SFB1292/2 TP20N author(s) upon reasonable request.
[Project Nr. 318346496]) and R.A.B. (BA 5939/2-1). B.E.C. and
R.A.B. are members of the Research Center for Immunotherapy Peer review: The peer review history for this article is available
(Forschungszentrum Immuntherapie, FZI) of the University Med- at https://publons.com/publon/10.1002/eji.202249925
ical Center Mainz.
Section 10: This work was supported by the German Research
Foundation (DFG): Ma1924/9-2
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