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.