Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues

Hans Christian Probst*, Patrizia Stoitzner*, Lukas Amon, Ronald A. Backer, Anna Brand, Jianzhou Chen, Björn E. Clausen, Sophie Dieckmann, Diana Dudziak, Lukas Heger, Katrin Hodapp, Florian Hornsteiner, Avi Hai Hovav, Lukas Jacobi, Xingqi Ji, Nadine Kamenjarin, Katharina Lahl, Imran Lahmar, Jelena Lakus, Christian H.K. LehmannDaniela Ortner, Marion Picard, Maria Paula Roberti, Lukas Rossnagel, Yasmin Saba, Carmen Schalla, Andreas Schlitzer, Barbara U. Schraml, Kristian Schütze, Anna Seichter, Kristin Seré, Athanasios Seretis, Sieghart Sopper, Helen Strandt, Martina M. Sykora, Hannah Theobald, Christoph H. Tripp, Laurence Zitvogel

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. 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 coauthors, making it an essential resource for basic and clinical DC immunologists.

Original languageAmerican English
Article number2249819
Pages (from-to)1-78
JournalEuropean Journal of Immunology
Volume53
Issue number11
DOIs
StatePublished - Nov 2023

Bibliographical note

Funding Information:
Sections 1.5 and Section 2.5 : We thank members of the Schraml lab for helpful discussions and critical reading of the manuscript. Work in the Schraml lab is supported by the European Research Council ‐ Starting Grant ERC‐2016‐STG‐715182 and the German Research Foundation ‐ Emmy Noether Grant Schr‐1444/1‐1, project‐ID 360372040–SFB 1335‐P08 and 322359157‐FOR2599‐A03.

Funding Information:
Section 1.7 : We would like to thank the whole team of the Laboratory for Langerhans Cell Research at the Department of Dermatology, Venereology & Allergology. This work was financially supported by the Austrian Science Fund (FWF) with the projects P‐21487‐B13, P‐27001‐B13, P‐33855‐B and DOC82. Additional funding was received from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska‐Curie grant agreement no 641549/Immutrain and by a PhD‐fellowship of the Austrian Academy of Sciences (OAW DOC/26015) to Florian Hornsteiner.

Funding Information:
Section 1.4 : We thank members of the Clausen and the Hovav laboratories for fruitful discussions and critical reading of the manuscript. Work in the Clausen laboratory is supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) to B.E.C. (CL 419/2‐2 [Project Nr. 315501751], CL 419/4‐1, CL 419/7‐1 [Project Nr. 503972215], and SFB1292/2 TP20N [Project Nr. 318346496]), and B.E.C. is a member of the Research Center for Immunotherapy (Forschungszentrum Immuntherapie, FZI) of the University Medical Center Mainz.

Funding Information:
Section 1.6 : This work was supported by the Confocal Microscopy Facility, a core facility of the Interdisciplinary Center for Clinical Research (IKZF) Aachen within the Faculty of Medicine at RWTH Aachen University.

Funding Information:
Section 1.2 : The Dudziak laboratory is thankful to its members for fruitful discussions. Parts of this methods paper are also included in the PhD thesis of Lukas Amon and Master's thesis of Lukas Jacobi. The Dudziak laboratory was supported by the German Research Foundation [Deutsche Forschungsgemeinschaft (DFG)] (CRC1181‐TPA7 261193037, DU548/5‐1, SFB TRR305 – B05, RTG2504‐401821119, RTG2599 421758891), the Interdisziplinäres Zentrum für klinische Forschung (IZKF) (IZKF‐A80, IZKF‐A87) and the Bavarian State Ministry of Science and Art (Bayresq.Net‐IRIS). Laurence Zitvogel and Diana Dudziak were co‐funded by the Agence Nationale de la Recherche (ANR, Ileobiome ‐ 19‐CE15‐0029‐01) and the DFG (DU548/6‐1).

Funding Information:
Section 1.1 and Section 2.1 : The Probst laboratory was supported by the German Research Foundation [Deutsche Forschungsgemeinschaft (DFG)] (SFB TR156‐B02, CRC1292‐TP13).

Funding Information:
Section 2.4 : We thank members of the Clausen and the Hovav laboratories for fruitful discussions and critical reading of the manuscript. Work in the Clausen laboratory is supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) to B.E.C. (CL 419/2‐2 [Project Nr. 315501751], CL 419/4‐1, CL 419/7‐1 [Project Nr. 503972215], and SFB1292/2 TP20N [Project Nr. 318346496]), and B.E.C. is a member of the Research Center for Immunotherapy (Forschungszentrum Immuntherapie, FZI) of the University Medical Center Mainz.

Funding Information:
Section 2.2 : The Dudziak laboratory is thankful to its members for fruitful discussions and to the Core Unit for cell sorting and immunomonitoring (FAU Erlangen). Parts of this methods paper are also included in the PhD thesis of Lukas Amon and Master's thesis of Lukas Jacobi. The Dudziak laboratory was supported by the German Research Foundation [Deutsche Forschungsgemeinschaft (DFG)] (CRC1181‐TPA7 261193037, DU548/5‐1, SFB TRR 305 ‐ B05, RTG2504‐401821119, RTG2599 421758891), the Interdisziplinäres Zentrum für klinische Forschung (IZKF) (IZKF‐A80, IZKF‐A87) and the Bavarian State Ministry of Science and Art (Bayresq.Net‐IRIS). Laurence Zitvogel and Diana Dudziak were co‐funded by the Agence Nationale de la Recherche (ANR‐ Ileobiome ‐ 19‐CE15‐0029‐01) and the DFG (DU548/6‐1).

Publisher Copyright:
© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.

Keywords

  • Dendritic cells
  • Discrimination of dendritic cell subsets
  • Mouse nonlymphoid tissue
  • Multiplex flow cytometry analysis
  • Tissue digestion protocols

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