Cls type 1

Isolation of the SVF and subsequently of ASC was performed as previously described [25 (link)]. Briefly, tissue samples were washed in PBS and cut into small pieces after removing blood vessels and connective tissue. Dissected tissues were incubated for 90 min at 37°C in a digestion buffer consisting of 200 U/ml collagenases (CLS Type I, Worthington Biochemical Corp., Lakewood, NJ) and 2% w/v BSA in PBS. After several purification steps, including filtration, centrifugation and incubation in erythrocyte lysis buffer (0.155 M Na₄Cl, 5,7 mM K₂HPO₄, 0.1 mM EDTA, pH 7.3), SVF was suspended in ASC2 medium. SVF cells were directly subjected to flow cytometric analysis or sorting, or for standard ASC isolation SVF cells were seeded in high density; after 16 h medium was changed to ASC1 medium, and cells were cultured in serum-free medium for 6 d. Then, ASCs were harvested, reseeded and cultured in PM4 medium (DMEM/F-12 medium with HEPES and L-glutamine containing 33 µM biotin, 17 µM pantothenate, 10 ng/mL EGF, 1 ng/mL FGF, 500 ng/mL insulin and 20 µg/mL ciprofloxacin, supplemented with 2.5% FBS) for amplification and used for experiments or frozen and stored in liquid nitrogen.