Isolation and Differentiation of Murine White Adipose Stromal Vascular Fraction Cells

Primary white adipose SVF cells were cultured as we described previously (Shan et al., 2016 (link)). Briefly, the inguinal fat pad was collected from 6-week-old female mice and washed with PBS twice. Then, the fat pad was minced with scissors and digested with collagenase type I (1.5 mg/ml, #SCR103, Sigma-Aldrich) at 37°C for 40 min. When the digestion was finished, the growth medium contained 85% high glucose DMEM medium (#11965126, Thermo Fisher Scientific) and 15% fetal bovine serum (#10099141, Thermo Fisher Scientific) was added to dilute the collagenase. The tissue debris was removed through a 70-μm cell strainer. The medium was subjected to centrifuge to get SVF cells pellet. SVF cells were resuspended with the growth medium. When the cells reached 90% confluence, they were induced to adipogenesis, with a cocktail containing DMEM, 10% fetal bovine serum, 2.85 mM recombinant human insulin (#I8830, Solarbio), 0.3 mM dexamethasone (#D8040, Solarbio), and 0.63 mM 3-isobutyl-methylxanthine (#I7018, Sigma-Aldrich). After 4 days, the cocktail was switched to a DMEM medium supplemented with 10% fetal bovine serum, 10 nM triiodothyronine (T3, #T6397, Sigma-Aldrich), and 200 nM insulin to induce mature adipocytes.