3t3 l1 pre adipocytes

Butyric acid and FFA-free bovine serum albumin (BSA) were purchased from Wako Chemicals (Osaka, Japan). All other chemicals were obtained from Nacalai Tesque (Kyoto, Japan). RAW264.7 mouse macrophages and 3T3-L1 mouse pre-adipocytes (American Type Culture Collection, Manassas, VA) were cultured in DMEM (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) with 292 μg/mL L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS) at 37 °C under 5% CO₂. Differentiation of 3T3-L1 pre-adipocytes was induced by addition of 0.5 mmol/L 13-isobutyl-1-methylxanthine, 2.5 μmol/L dexamethasone, and 10 μg/mL insulin (Wako Chemical) in DMEM containing 10% FBS, beginning 2 days after the cells reached confluence in a 24-well plate. The medium was then replaced with DMEM containing 10% FBS and 5.0 μg/mL insulin and was changed every 2 or 3 days. Twenty days after induction of differentiation, the hypertrophied 3T3-L1 adipocytes were used in experiments. Cell viability was assessed using an MTT cell viability assay kit (R&D Systems, Minneapolis, MN).