Isolation and Differentiation of Mouse Adipose-Derived Stromal Cells

Stromal-vascular fractions (SVF) were isolated as previously described [5 (link),12 (link)]. Briefly, inguinal white adipose tissues were dissected from 8-week-old mice, minced and digested in 1.5 %BSA KRBH containing 1 mg/ml collagenase I (Solarbio, China) at 37 °C for 1h. Digested tissue was filtered through 100 μm nylon mesh and centrifuged to separate floating adipocytes. The resulting pellet (SVF) was cultured in DMEM, supplemented with 10 % FBS, 1 % MEM non-essential Amino acid solution (11140050, Gibco), 1 % GlutaMax (35050061, Gibco) and 100U/ml penicillin and streptomycin (growth medium). Adipogenesis was induced by incubation in growth medium supplemented with 1x ITS (Gibco), 1 μM dexamethasone, 33 μM Biotin, 2 nM T3, 17 μM Pantothenate, 0.5 mM IBMX and 1 μM rosiglitazone (differentiation medium). After 2–4 days, the medium was changed to differentiation medium without IBMX and rosiglitazone (maintenance medium), and then changed again every 2 days until full differentiation (i.e., day 8–10). For activin B treatments, matured adipocytes were serum starved for 2h and then incubated with indicated concentration of activin B for either 20min for detection of SMAD phosphorylation levels, or 24h for RNA extraction. For CL-316,243 treatment, matured adipocytes were incubated with 10 μM CL-316,243 for 20min before harvested for protein extraction.