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. We are no longer limited by complex instrumentation, but by our creativity to ask the critical questions. 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.
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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. 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. 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.įor 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. 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. 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. The main strengths of this technology, i.e. 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.
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. 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. 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. 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.
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.
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. 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. The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. Introduction: Guidelines for the use of flow cytometry in immunology