Brown adipocyte cell line was established by immortalization of preadipocytes obtained from interscapular BAT of mice as previously described [18 (link)]. Immortalized preadipocytes were cultured in growth medium (Dulbecco’s modified Eagle’s medium (Welgene, South Korea) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Thermo Fisher, USA)) at 37 °C in a humidified atmosphere with 5% CO2. For adipocyte differentiation, confluent preadipocytes were exposed to differentiation medium containing 2.5 mM of isobutylmethylxanthine (IBMX, Cayman, USA), 1 μM of dexamethasone (Cayman, USA), 10 μg/mL of insulin (Sigma, USA), 125 μM of indomethacin (Cayman, USA) and 1 nM of triiodothyronine (T3, Cayman, USA) for 3 days followed by maintenance medium containing insulin (10 μg/mL) and T3 (1 nM) for 3 days.
C3H10T1/2 (ATCC, USA) cells were cultured in growth medium. For adipogenic differentiation, C3H10T1/2 cells were exposed to DMEM containing 20 ng/mL of bone morphogenetic protein 4 (BMP4, R&D systems, USA) for 2 days, differentiated in differentiation medium for 3 days, and exposed to maintenance medium for 3 days.
C3H10T1/2 (ATCC, USA) cells were cultured in growth medium. For adipogenic differentiation, C3H10T1/2 cells were exposed to DMEM containing 20 ng/mL of bone morphogenetic protein 4 (BMP4, R&D systems, USA) for 2 days, differentiated in differentiation medium for 3 days, and exposed to maintenance medium for 3 days.