Primary brown preadipocytes isolation and differentiation

Primary brown preadipocytes were isolated from 4-week–old WT mice as described previously [69]. Adipocyte differentiation was induced by DMEM medium containing 10% FBS, isobutylmethylxanthine (0.5 mM), indomethacin (125 nM), dexamethasone (0.5 μM), insulin (850 nM), T3 (1 nM), and rosiglitazone (0.5 μM). Two days after induction, cells were switched to maintenance DMEM medium containing 10% FBS, insulin (850 nM), T3 (1 nM), and rosiglitazone (0.5 μM). The adipocytes were fully differentiated after 6–7 days of induction.