F4 80 apc cy7

Stromal vascular fraction (SVF) was obtained from pgWAT by treatment with 2 mg/mL collagenase (Sigma) for 45 min at 37 °C. The isolated SVF was resuspended in cold Hank’s balanced salt solution (HBSS) with 2% fetal bovine serum (FBS). Cells were incubated with CD45-PE-Cy7 (eBiosciences), F4/80-APC-Cy7 (BioLegend), CD206-Alex647 (Serotec, Inc.) and CD11c-PE (BD Pharmingen) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Sytox Blue (Thermo Scientific). Cells were analyzed on a BD FACSAria cell sorter after selection by forward scatter and side scatter, followed by exclusion of dead cells with Sytox Blue staining, and analyzed for cell-surface markers using FlowJo software (Tree Star). M1 or M2 macrophages were identified as F4/80-positive/CD11c-positive/CD206-negative or F4/80-positive/CD11c-negative/CD206-positive cells, respectively. The data are shown as the percentage of M1 and M2 macrophages. For sorting preadipocytes, endothelial cells and macrophages, cells were incubated with CD31-PE-Cy7 (eBiosciences), F4/80-APC (eBiosciences), and Sca-1 FITC (eBiosciences) antibodies for 30 min in HBSS containing 2% FBS on ice and then washed and resuspended in solution with Propidium Iodide Staining Solution (Sigma). Gating strategies are presented in Supplementary Figure 12 and Supplementary Figure 13.