Strategies for flow-cytometric analysis of intracellular IL17-A, IFNγ and FoxP3. Peripheral blood lymphocytes were incubated for 4 hours with PMA/Ionomycin/BFA (IL17 and IFNγ) or in medium alone (FoxP3) followed by intracellular staining and analysis by flow cytometry. A. IFNγ and IL17-A accumulation in CD8- (referred to as CD4+ cells) after 4 hours of stimulation with PMA/Ionomycin/BFA. The detection of IL17-A in CD8- (CD4+) T cells was verified by using two different antibodies. B. The levels of regulatory T cells were analyzed by detection of CD4+ CD25+ FoxP3+ cells. For both panels CD3+ CD8+ T cells were used as negative intra-sample controls for both TH17 and TREG cells.