Heat inactivated bovine serum

Adipose tissue from relevant depot harvested were minced in Krebs buffer (described above) supplemented with 1% bovine serum and 0.5 mg/ml collagenase I (Themo Scientific) and dispase (2.4 U/ml) (Sigma) following 45 min incubation at 37 °C. Minced tissues were then washed with Hank’s buffered salt solution (HBSS), supplemented with 10 mM HEPES (GIBCO) and 2% heat-inactivated bovine serum (Corning). Following centrifugation steps (2000 rpm 5 min), the pellet was resuspended in red blood cell lysis buffer (155 mM NH4CL, 12 mM NaHCO3, 0.1 mM EDTA) for 5 min. Following additional washing steps with HBSS, stromal vascular fraction was obtained and used for downstream analysis. For analysis of populations, the following antibodies were used: for removal of lineage positive cells: TER-119-PB (1:100), CD31-PB (1:100), CD45-PeCy7 (1:100). For isolation of preadipocytes, lineage negative cells were then selected for the expression of the following antibodies: Sca1- APC Cy7 (1:100), CD34-FITC (1:100), CD29-PE (1:200), IL6-APC (1:100) All monoclonal antibodies were from BD Biosciences or eBioscience. The LSRII cell analyzer was used for data acquisition, sorting was done using AriaI. All data analysis was performed using the FlowJo software.