Isolation and Differentiation of Murine Adipose SVF Cells

SVF cells from 6- to 7-week old male C57BL/6J mice were isolated as described previously^{5 (link)}. In brief, adipose tissue was minced on ice and digested with 10 mg/ml collagenase D (Roche) and 2.4 mg/ml dispase II (Roche) in phosphate-buffered saline (PBS) supplemented with 1% penicillin/streptomycin for 45 min at 37 °C, followed by quenching with complete medium and filtering through a 100 μm strainer (BD Biosciences). The cell suspensions were centrifuged, suspended and filtered through a 40 μm strainer (BD Biosciences) and then further centrifuged and suspended before plating onto 10 cm dishes. SVF cells were cultured in DMEM/F12 supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). Adipocyte differentiation was carried out by treating confluent cells in growth medium supplemented with 0.5 mM IBMX, 125 nM indomethacin, 1 μM dexamethasone, 850 nM insulin, and 1 nM T3 for 48 h. Following this, the cells were maintained in growth medium supplemented with insulin and T3 for 8 days. To analyze the effects of BBR on differentiation, BBR, or rosiglitazone (a positive control) was added throughout the differentiation. Experiments were performed on day 8 of differentiation.