Flow cytometry sorting and analysis of Th subtype populations. (A) FACS gating strategies used to sort Th subtypes after growth in polarizing conditions. Initial gates selected for singlet lymphocyte events and were followed by sorting for specific cell surface markers as follows. Th1: CXCR3+, PI−, depletion markers− (CD11b−CD11c−Ly6G−CD8a−CD19−). Th2: CD4+, PI−, depletion marker−. Th17: CCR6+, Cd8a−, PI−. iTreg: GFP+ PI−. (B) Verification of CD4+ cell lineage identities by intracellular flow cytometry staining for the factors indicated. Cells were analysed using fluorescently-labelled antibodies against the indicated markers. Th1, Th2 and Th17 cells were restimulated prior to analysis as described in Methods. Percentages within the quadrants/gates are indicated, and are representative of the purities routinely obtained.