Flow cytometry.
For analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL). For cell surface marker staining, cells were incubated for 20 min at 4degreesC in staining buffer with the following antibodies: anti-CD152-PE (CTLA-4; BD), anti-PD-1 (eBiosciences), anti-GITR (R & D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter). The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a. For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25. Anti-FcgammaRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding. To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting. Dead cells were excluded with 4'',6-Diamidino-2-phenylindole (DAPI). To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells. All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences. 
