2.4. Isolation of the stromal vascular fraction (SVF) from adipose

SubQ and EF tissue was aseptically excised from C57BL/6J mice, minced, and incubated in basal medium (Zenbio, Research Triangle Park, NC) supplemented with collagenase (1 mg/mL) and penicillin (100 U/mL)/streptomyocin (100 µg/mL) for 1 hour with shaking at 37°C, as previously described in 12-well plates at a seeding density of 50,000 cells/cm² [21]. Two days after SVF cells had reached 100% confluence (day 0), the medium was changed to differentiation media and then replaced every other day with fresh media for 8 days. Cells were harvested at day 8 after differentiation for ORO staining and RNA isolation using Maxwell RSC simplyRNA Cell Kit; cDNA synthesis and real-time PCR were performed as described below.