Adipocyte Fractionation and Differentiation

Stromovascular cells (SVC) and adipocytes from eWAT were fractionated, as previously described [21 (link), 26 (link)]. For gene expression analysis, dissociated adipose tissue was fractionated by magnetic cell sorting (MACS) with anti-PDGFRα-PE/anti-PE-microbeads, anti-F4/80-FITC/anti-FITC-microbeads, and anti-CD31-APC/anti-APC-microbeads (Miltenyi Biotech). For adipogenic differentiation, MACS-isolated PDGFRα was expanded in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, and then differentiated by DMEM supplemented with a standard adipogenic cocktail for 7 days, as described previously [25 (link)]. To determine levels of adipogenic differentiation, differentiated cells were labeled with 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY 558/568 C12) (Invitrogen Molecular Probes) or Oil Red O (Sigma-Aldrich). Mitochondrion-labeling in live cells was performed using red-?uorescent mitochondrion-selective probe MitoTracker Red CMXRos (Thermo Fisher, Waltham, MA, USA).