Be0055

Spleen and lymph nodes were obtained from 6-week-old to 8-week-old B6 Foxp3^GFP or BALB/c Foxp3^GFP mice and a single cell suspension was prepared. Red blood cells were removed using a 1× ACK lysis buffer (Gibco). CD4 cells were isolated by depleting CD25⁺ cells using the CD25 MicroBead Kit (Miltenyi Biotec) and subsequent enrichment of CD4 T cells using CD4 T Cell Isolation Kit (Miltenyi Biotech). Cells were cultured in complete Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) with 1% non-essential amino acids (NEAA), 1% Penicillin-Streptomycin, 100 IU/mL L-glutamine and 10% Fetal Bovine Serum (FBS) (Gibco). The complete media was supplemented with 1× β-Mercaptoethanol (Thermo Fisher Scientific), 5 ng/mL of TGF-β (240-B-010, R&D Systems), 20 ng/mL of IL-2 (402 ML-020, R&D Systems), 10 µg/mL of anti-IFN-γ (BE0055, InvivoGen), 10 µg/mL of anti-IL-4 (BE0045, InvivoGen) and 1 µg/mL anti-CD28 (16-0281-86, Thermo Fisher Scientific) and cultured in 5 µg/mL anti-CD3 (16-0031-85, Thermo Fisher Scientific) coated plates at a concentration of 1×10⁶ cells/mL. On day 3, cells were cultured in RPMI medium with 20 ng/mL IL-2. Cells were harvested on day 7 for studies. Over 90% of iT_reg expressed CD25 and GFP.