Flow cytometry is a powerful technique that allows simultaneous detection of multiple markers on a specific cell population. This method is virtually unlimited as long as the specimen of interest can be put into a single-cell suspension for staining and subsequent analysis by the flow cytometer. Most investigators using this methodology are doing so because their cell population is rare in frequency and requires multiple markers to characterize their population of interest; thus standard methods such as Western blot and IHC are unsuitable due to limitations in cell number and the number of markers available. Most investigators using this method are using 6-14 parameters to study their cell populations of interest: however, using a large number of fluorochrome-labeled antibodies is hampered by the fact that suboptimal fluorochromes must be used, and that high and low cell density markers must be chosen with care. This is further complicated when the cell markers of interest are cytokines, transcription factors, surface markers, and/or phosphorylated proteins, each potentially requiring a specialized buffer system for optimal detection of the antibody of interest. This chapter focuses on optimizing flow cytometry staining methods for simultaneous detection of surface markers, transcription factors, secreted cytokines, and phosphorylated antibodies in a single stain on CD4+ human Th2 cells.
Keywords: CCR3; CD4; Flow cytometry; GATA-3; IL-10; Peripheral blood mononuclear cells (PBMCs); Phospho-STAT6; Phospho-antibodies; T helper 2 cells (Th2); Th2 cytokines.