Flow cytometry is a powerful and robust technology for detecting and monitoring multiple markers at the level of single cells. Since its early development, flow cytometry has been used to assess heterogeneity in a cell suspension. Over the years, the increasing number of parameters that could be included in a single assay combined with physical separation by fluorescence-activated cell sorting (FACS) revealed that the T cell compartment is extremely heterogenous in terms of phenotypic diversity, functional capacity, and transcriptional regulation. While naïve T cells are fairly homogenous, diversity becomes extreme in the antigen-experienced memory compartment. The precise identification of memory subsets by the simultaneous analysis of multiple markers by flow cytometry is key not only to basic science but also for the correct immunomonitoring of patients undergoing immunotherapy or for T cell-based therapeutic approaches. In this chapter, we provide guidelines to optimize complex flow cytometry panels, to achieve correct fluorescence compensation and determine positivity for a given antigen. Correct selection of reagents and their validation is essential to the success of the assay. Despite having been developed for the identification of human naïve and memory T cell subsets, the concepts illustrated here can be applied to any experiment aiming to investigate n parameters by flow cytometry.
Keywords: Antibody; Cell sorting; Compensation; FACS; Memory T cells; Naïve T cells; Polychromatic flow cytometry; Reagent titration; T cell differentiation.