2.5. Primary cell isolation

For SVF preparation, adipose tissue subcutaneous (inguinal), visceral (perigonadal), and brown (interscapular) fat pads were dissected, finely minced, and incubated with collagenase (1 mg/ml) in collagenase buffer (25 mM NaHCO3, 12 mM KH2PO4, 1.2 mM gSO4 1.2 mM, 4.8 mM KCl 120 mM NaCl, 1.4 mM CaCl2 5 mM Glucose, BSA 2.5%, pH = 7.4) for 1 h at 37 °C. Cells were washed in DMEM, 10% FBS and filtered through 40 μM cell strainers; erythrocytes were lysed by washing in erythrocyte lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) and plated in DMEM, 10% FBS, 1% penicillin/streptomycin.