Cell culture
Sorted CD4+GFP+ Treg cells or CD4+GFP- T cells (2 × 105 cells/well on 96-well flat-bottom plate) were cultured in RPMI 1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS), 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 50 µM 2-mercaptoethanol. The sorted cells were cultured in the presence PMA 50 ng/mL, ionomycin 1 µL/mL, and Golgistop 1 µL/mL in a flat-well for 8 h and analyzed using cytokine staining.