Isolation and Culture of Adipose-Derived Cells

Adipose tissue biopsies of the abdominal subcutaneous region were performed under local anesthesia [35 (link)]. Adipose biopsies were taken in the overnight fasted state between 8 and 10 AM. The scWAT was digested with collagenase (type 1, 1 mg/2ml in HBSS) for 30-60 min at 37° C with shaking (100rpm), as described previously [36 (link)]. The mixture was passed through a 250-micron mesh, centrifuged at 500g for five minutes and floating adipocytes were discarded. Cell pellets were treated with erythrocyte lysis buffer (BioLegend, Cat: 420301) for five to 10 minutes at room temperature followed by centrifugation at 500g for five minutes. After discarding the supernatant, cells were resuspended in proliferation media (PM) (Gibco α-Minimum Essential Media (αMEM), 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin), then plated and cultured in an incubator at 37° C at 5% carbon dioxide. Media was replaced after four hours to remove blood cells. APCs continue to grow with PM changes every other day until they reached ~80% confluency, then they were frozen in freezing media (10% FBS, 10% DMSO in αMEM) and stored in dewars containing liquid nitrogen.