Two-color flow cytometry was performed as described previously (33, 37). Briefly, 1 × 106 splenocytes suspended in flow staining buffer were incubated at 4°C with appropriately diluted fluorescein isothiocyanate (FITC)–labeled antibody to CD4 for 30 min, washed, and resuspended in fixation and permeabilization solution. After incubation in the dark for 30 min, cells were washed, blocked with test Fc block (anti-mouse CD16/32) in permeabilization buffer, and subsequently incubated with appropriately diluted phycoerythrin-labeled antibodies to Foxp3 at 4°C in the dark. After incubation, the cell suspension was centrifuged, washed thrice, and resuspended in flow staining buffer. The cells were then analyzed through FACS (BD Biosciences, San Jose, CA). Cells were gated on the basis of morphological characteristics. Apoptotic and necrotic cells were not accepted for FACS analysis.

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