Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

Grants

Funding Acknowledgements

Hyun-Dong Chang is supported by the Dr. Rolf M. Schwiete Foundation. Susanne Melzer and Attila Tarnok thank De Novo Software for providing FCS Express. Enrico Lugli is supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC IG20676). Gabriele De Simone and Giovanni Galletti were supported by Fellowships from the Fondazione Italiana per la Ricerca sul Cancro-Associazione Italiana per la Ricerca sul Cancro (FIRC-AIRC). Jun Dong is supported by Deutsche Forschungsgemeinschft (DFG, German Research Foundation) Projektnummer 389687267 and Chinesisch-Deutsches Zentrum fur Wissenschaftsforderung [Sino-German Center for Research Promotion (SGC)] grant C-0072. Nicola Gagliani, Samuel Huber and Franziska Muscate are supported by DFG fundings: SFB841,GA 2441/3-1, HU 1714/10-1. The tetramer APC-conjugated H-2K (d) HIV-1 gag<INF>197-205</INF> AMQMLKETI used in TDS assay for mouse blood T cells was obtained through the NIH Tetramer Facility. Larissa Nogueira Almeida was supported by DFG research grant MA 2273/14-1. Supported by the following grants: AIRC 5X1000 2018 id. 21147 (Lorenzo Moretta); AIRC IG 2017 id. 19920 (Lorenzo Moretta); RC-2020 OPBG (Lorenzo Moretta); AIRC and European Union's Horizon 2020 research and innovation program under the Marie Skodowska-Curie grant agreement No 800924 (Linda Quatrini). Dirk Baumjohann was supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Emmy Noether Programme BA 5132/1-2 (252623821) and Germany's Excellence Strategy EXC2151 (390873048). The following formed the writing committees for the indicated sections: A. Graham Pockley, Gemma A. Foulds, Gabriele Multhoff (I.1Essential Controls and Reproducibility in Flow Cytometry); Susanne Melzer, Attila Tarnok (I.2 MIFlowCyt compliance and data display of flow cytometry data in immunological research); Florian Mair, Claudia Haftmann (I.3. Panel design in high-dimensional flow cytometry); Sara De Biasi, Lara Gibellini, Andrea Cossarizza (II. Rare cells - General rules); Samuele Notarbartolo, Daniela Latorre, Antonino Cassotta, Chiara Vasco, Federica Sallusto, Sergio Abrignani, Jens Geginat (III.1. Human conventional alpha beta CD4<SUP>+</SUP> T cells); Regina Stark, Kylie Quinn, Jessica Borger (III.2 Murine Conventional alpha beta CD4<SUP>+</SUP> T cells); Jasper J. P. van Beek, Gabriele De Simone, Giovanni Galletti, Enrico Lugli (III.3 Human conventional alpha beta CD8 T cells); Regina Stark, Kylie Quinn, Jessica Borger (III.4 Murine Conventional alpha beta CD8 T cells); Jun Dong (III.5 Human tissue resident memory T cells); Regina Stark, Klaas van Gisbergen (III.6 Murine tissue resident memory T cells); Jose Antonio Garrote, Aida Fiz, David Bernardo, Fernando Gabriel Chirdo, Eduardo Arranz (III.7 Human intestinal intraepithelial T cells); Immo Prinz, Joana Barros-Martins (III.8 Mouse intestinal intraepithelial T cells); Marcello Pinti (III.9 Immune senescence (aging) of human T cells); Luka iin-Sain, Katarzyna M. Sitnik, Kylie M. Quinn (III.10 Immune senescence (aging) in murine T cells); Dominic A Boardman, Eleni Christakou, Laura Cook, Michael Delacher, Markus Feuerer, Qing Huang, Sabine M Ivison, Megan K Levings, Katherine N. MacDonald, Timothy I.M. Tree, Kirsten A Ward-Hartstonge, Jennie H. M. Yang (III.11 Human FOXP3+ regulatory T cells); Michael Delacher, Juhao Yang, Jana Niemz, Lieke Sanderink, Markus Feuerer, Jochen Huehn (III.12 Murine Foxp3+ regulatory T cells); Chiara Vasco, Nadia Pulvirenti, Paola Gruarin, Jens Geginat (III. 13 Human IL-10 producing regulatory T cells (Tr1 cells)); Franziska Muscate, Leonie Brockmann, Samuel Huber, Nicola Gagliani (III.14 Murine Tr1 cells); Anouk von Borstel, Martin S. Davey (III.15 Unconventional human T cells: gamma delta (gamma delta) T cells); Immo Prinz, Anneke Wilharm, Inga Sandrock (III.16 Unconventional murine T cells: gamma delta T cells); Christopher M. Harpur, Dale I. Godfrey, Garth Cameron (III.17 Unconventional human T cells: NKT cells); Andreas Krueger, Heike Kunze-Schumacher, Nikita A. Verheyden (III.18 Unconventional murine T cells: NKT cells); Garth Cameron, Nicholas A. Gherardin, Alexandra J. Corbett, Sidonia B. G. Eckle, James McCluskey, Dale I. Godfrey, Hui-Fern Koay (III.19 Unconventional human T cells: mucosal-Associated Invariant T (MAIT) cells); Andreas Krueger, Heike Kunze-Schumacher, Nikita A. Verheyden (III.20 Unconventional murine T cells: MAIT cells); Carina Saggau, Alexander Scheffold, Petra Bacher (IV.1 Antigen-specific T cell cytometry: Functional read-outs); Anastasia Gangaev, Steven Ketelaars, Pia Kvistborg (IV.2 Measuring antigen-specific CD8 T cell responses); Immanuel Andra, Dirk Busch, Matthias Schiemann, Kilian Schober (IV.3 Antigen-specific T cell cytometry: MHC multimers); Alessio Mazzoni, Laura Maggi, Francesco Annunziato (IV.4 Identification of SARS-CoV-2 specific human CD4+ T cells by 8-color flow cytometry); Dirk Baumjohann, Yinshui Chang, Johanna E. Huber (IV. 5 Adoptive T cell transfers as a read-out for Ag-specific immune responses in mice); Maria B. Rodrigo, Nina Kessler, Laura Knop, Natalio Garbi, Thomas Schuler (IV.6 Cytotoxicity); Alessio Mazzoni, Laura Maggi, Francesco Annunziato (IV.7 Measurement of signal transduction pathways in human T cells); Alessio Mazzoni, Laura Maggi, Lorenzo Cosmi (IV.8 Live cytokine-producing cell sorting with cytokine secretion assay<SUP>TM</SUP>); Laura Maggi, Alessio Mazzoni, Francesco Liotta (IV.9 Quantification of soluble cytokines with cytometric bead array); James Wing, Ilenia Cammarata, Vincenzo Barnaba, Shimon Sakaguchi (IV.10 Treg suppression assays); Ambra Natalini, Sonia Simonetti, Giovanna Peruzzi, Miguel Munoz-Ruiz, Leticia Monin, Irma Pujol-Autonell, Hefin Rhys, Heather M. Long, Adrian C. Hayday, Francesca Di Rosa (IV.11 Measurement of T cell proliferation by the T<INF>DS</INF> assay); Annika Wiedemann, Andreia Lino, Thomas Dorner (V.1 Human B cells and their subsets); Annika Wiedemann, Andreia C. Lino, H. Kristyanto, Hans U. Scherer, Thomas Dorner (V.2 Human antigen-specific B cells); Willem van de Veen, Oliver Wirz, Mubeccel Akdis (V.3 Human immunoglobulin heavy chain isotypes); Oliver Wirz, Willem van de Veen, Mubeccel Akdis (V.4 Human regulatory B cells); Larissa Nogueira Almeida, Rudolf Armin Manz (V.5 Murine B cells and their subsets, including Bregs); Antonia Niedobitek, Axel R. Schulz, Marie Burns, Julia Schulze, Henrik E. Mei (V.6 Human antibody-secreting plasmablasts and plasma cells); Katharina Kriegsmann, Leo Hansmann, Calin Manta, Michael Hundemer (V.7 Human plasma cells in multiple myeloma); Sebastian Schulz, Katharina Pracht, Jens Wittner, Dirk Mielenz, Wolfgang Schuh, Hans-Martin Jack (V.8 Murine antibody-secreting plasmablasts and plasma cells); Franziska Szelinski, Annika Wiedemann, Andreia C. Lino, Thomas Dorner (VI.1 Measurement of signal transduction pathways in human B cells); Simon Fillatreau, Rudolf Armin Manz, Guido Heine (VI.2 Live cytokine-producing human B cell sorting with secretion assay: the case of IL-10); Baerbel Keller, Marie Follo, Klaus Warnatz (VI. 3 Antigen-receptor mediated Ca2+ mobilization in lymphocytes); Dirk Baumjohann, Yinshui Chang, Johanna E. Huber (VI.4 Adoptive B cell transfers as a read-out for Ag-specific immune responses in mice); Daniela Carolina Hernandez, Christina Stehle, Oliver Hoelsken, Andreas Diefenbach, James Di Santo, Chiara Romagnani (VII.1 Human Innate Lymphoid Cells); Christina Stehle, Daniela Carolina Hernandez, Oliver Hoelsken, Andreas Diefenbach, James Di Santo, Chiara Romagnani (VII.2 Murine Innate Lymphoid Cells); Linda Quatrini, Lorenzo Moretta, Genny del Zotto (VIII.1 Human NK cells); Linda Quatrini, Lorenzo Moretta, Genny del Zotto (VIII.2 Murine NK cells); Regine J. Dress, Amit A. Patel, Charles-Antoine Dutertre, Florent Ginhoux (IX.1 Human mononuclear phagocytes: Monocytes, macrophages, and dendritic cells); Regine J. Dress, Amit A. Patel, Charles-Antoine Dutertre, Florent Ginhoux (IX.2 Murine mononuclear phagocytes: Monocytes, macrophages, and dendritic cells); Regine J. Dress, Amit A. Patel, Charles-Antoine Dutertre, Florent Ginhoux (X.1 Human mononuclear phagocytes: Monocytes, macrophages, and dendritic cells); Regine J. Dress, Amit A. Patel, Charles-Antoine Dutertre, Florent Ginhoux (X.2 Murine mononuclear phagocytes: Monocytes, macrophages, and dendritic cells); Janina Schoen, Christine Schauer, Jeeshan Singh, Jasmin Knopf, Luis Enrique Munoz, Martin Herrmann (XI.1 Neutrophil, eosinophil, and basophil phenotypes); Janina Schoen, Christine Schauer, Jeeshan Singh, Jasmin Knopf, Luis Enrique Munoz, Martin Herrmann (XI.2 Human bone marrow and cord blood neutrophils); Janina Schoen, Christine Schauer, Jeeshan Singh, Jasmin Knopf, Luis Enrique Munoz, Martin Herrmann (XI.3 Murine bone marrow neutrophils); Frank A. Schildberg (XII.1 Human bone marrow stromal cells); Rebecca Cornelis, Richard Addo, Daniel Lenz, Mir-Farzin Mashreghi (XII.2 Murine bone marrow stromal cells); Peter K. Jani, Julia Tornack, Fritz Melchers, Gulce Itir Percin, Julia Frobel, Claudia Waskow (XIII Hematopoietic stem cell phenotypes); Jenny F. Kuehne, Christine S. Falk (XIV Tumor cell phenotypes); Lennard Ostendorf, Chotima Bottcher, Sarah Starossom, Katja Kobow, Pascale Eede, Helena Radbruch (XV Brain and neural cell phenotypes); Laura J. Pallett, Mala K. Maini, Matteo Iannacone (XVI.1 Liver cell phenotypes); Katrin Neumann, Francesco Andreata, Cristian G. Beccaria, Matteo Iannacone (XVI.2 Murine liver cells).