Isolation of Stromal Vascular Fraction Cells

Stromal vascular fraction (SVF) cells were isolated by a previously described methodology^{20 (link)}. Briefly, small pieces of adipose tissue (1–2 g of SAT or MAT) (avoiding blood vessels) were added to tubes containing Hanks’ balanced salt solution supplemented with 4% BSA, 10 mM HEPES and Liberase™ TL Research Grade (Roche Diagnostics, Risch-Rotkreuz, Switzerland) and incubated in a water bath up to 60 min at 37 °C, with manual shaking each 10 min. After water bath incubation, digested samples were homogenized to single-cell suspensions, passed through a 100-μm cell strainer (BD Biosciences Pharmingen, San Diego, CA) and centrifuged at 280 × g for 10 min at 4 °C. Cells at the bottom, corresponding to the SVF were resuspended in Dulbecco’s PBS, supplemented with 2% FBS (Biowest, Nuaillé, France), 2 mM EDTA and 10 mM HEPES (all from Sigma-Aldrich) for flow cytometry studies and complete RPMI medium for cell sorting experiments.