Guidelines for the use of flow cytometry and cell sorting in immunological studies

The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy‐to‐use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system. However, the moon has a dark side. The main strengths of this technology, i.e. the fact that it is relatively easy to use and that often only a brief training is sufficient to use a flow cytometer and start producing data, is also its main weakness. Indeed, in several (too many) papers, the eye of a well‐trained cytometrist can identify aspects that would need, to be polite, a “little” improvement. Not to mention the cases in which technical mistakes are performed, involving, among others, the use of (in)adequate controls, the (lack of appropriate) compensation, sorting strategies, or even the description of the methods used. For this reason, the editorial team of the European Journal of Immunology feels it is worthwhile to offer our community guidelines for the correct use of cytometric techniques in the field of immunology. Thus, starting at the European Congress of Immunology (ECI 2015) in Vienna (Austria) and under the guidance of Professor Andreas Radbruch, we asked colleagues and friends, all renowned in this field, to contribute by sharing their knowledge in their particular areas of expertise, in order to present a collection of protocols of great interest. Such information includes, among others, suggestions and tricks regarding how to study cell phenotypes, the type or amount of molecules produced or secreted after stimulation by a cell population of interest, signalling processes, differentiation, proliferation or cell death, cytotoxic activities, cell‐cell interactions, activity of intracellular organelles such as mitochondria, different types of response induced against tumours or by anticancer or immunosuppressive drugs, transcription factor activity, the quantification of soluble molecules, drug uptake, and rare events. Today's challenges also involve the choice of reagents, the preparation and eventual storage of the cells under analysis, the overall experimental plan and, last but not least, data analyses. We are no longer limited by complex instrumentation, but by our creativity to ask the critical questions. These “Guidelines for the use of flow cytometry and cell sorting in immunological studies” thus represent a community effort to collect the currently accepted best methods for monitoring most of the variation of the major players of immune system (along with their organelles and functionality) and include standards for data interpretation, as well as cautions about technical issues. One aspect of the guidelines concerns data reproducibility, a topic that has recently attracted considerable attention. Therefore, the guidelines are meant to help researchers avoid potential pitfalls that could drastically alter the interpretation of their data. While preparing the guidelines, feedback was received that we feel should be highlighted in this Introduction. Firstly, “FACS” (fluorescence activated cell sorting) should only be used for Becton Dickinson (BD) technologies as it is a BD trademark (FACSTM); the more general term “flow cytometry cell sorting” should be used to be company agnostic. Secondly, CD mAbs and not anti‐CD mAbs (in other words CD1 mAb and not anti‐CD1 mAb, for example) should be used. This is because the CD nomenclature is primarily a system to cluster/characterize mAbs and it was only later accepted to use this system to also describe the respective CD molecules. Thirdly, although the guidelines are as comprehensive as possible, there are naturally limitations e.g. only a subset of antibodies and antigens are shown and, at times, only certain reagents/companies are used as examples. It is our opinion that all efforts must be improved—this is how science works! Thus, we would be glad to receive from readers of the European Journal of Immunology critical comments, new ideas, and even suggestions for new articles for possible future updates of the Guidelines. Before closing, we would like to thank four people who played a major role in ensuring that Andreas Radbruch's and Andrea Cossarizza's vision became a reality. These are Hyun‐Dong Chang and Ute Hoffman, both at the DRFZ, and Karen Chu, former Associate Editor, and Cate Livingstone, Managing Editor of the European Journal of Immunology. Together this core team coordinated author invitations, and the submission, peer review and revision of all the sections and proofs, as well as ensuring that community feedback was sought and incorporated. We would also like to thank the full editorial team of the European Journal of Immunology for their invaluable work on this project.