Isolation of adipose tissue cell populations by FACS

Sorting of adipose tissue cell populations was performed as previously described (18). Briefly, adipocytes and SVF were separated by centrifugation of digested and strained adipose tissue. Floating adipocytes were lysed in QIAzol for further analysis, and the SVF was suspended in BSA buffer and incubated with Fc Block for 10 min on ice. To deplete the erythrocytes, the cells were incubated with Anti-Ter-119 MicroBeads (130-049-901, Miltenyi Biotec) for 15 min on ice and applied to an OctoMACS Separator according to the manufacturer’s instructions. The collected flow-through was stained for specific cell populations with CD45–fluorescein isothiocyanate (30-F11, eBioscience), CD31–eFluor 450 (390, eBioscience), CD29–peridinin chlorophyll protein complex–eFluor 710 (HMb1-1, eBioscience), CD34–Alexa Fluor 647 (RAM34, BD Biosciences), and Sca-1–Alexa Fluor 700 (D7, eBioscience) for 30 min on ice. Cells were washed, and sorting was performed on a BD FACSAria (BD Biosciences) by using unstained and FMO-stained as negative controls. Compensation was performed by single antibody-stained cells. Sorted cells were pelleted by centrifugation and lysed in QIAzol for further analysis.