Collagenase 2 and xi

Cells were isolated from adipose tissue by collagenase digestion according to De Clercq et al. [14] . Adipose tissue was minced and digested in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid phosphate buffer (HEPES, 2 mL/g of tissue) containing 2% BSA and 2 mg/mL collagenase II and XI (800 U/mg; Sigma) in a shaking water bath for 45 min at 37°C. A fivefold excess of collagenase buffer free was added before cells were centrifuged at 400 × g for 10 min to separate floating adipocytes from the pellet of stroma vascular (SV) cells.
After re-suspension in Dulbecco's Modified Eagle Medium (DMEM), SV cells were successively filtered through a 200-and 25-µm nylon mesh (Nytex, Dutscher Brumath, France). Cells were isolated from skeletal muscle according to Perruchot et al. [15] (link). Muscle was digested for one hour in a water bath in a mixture of trypsin 0.25% (Invitrogen, Cergy Pontoise, France)/Collagenase de type II 1.5 mg (PAA Les Mureaux, France)/DNAse 0.1% Sigma Saint-Quentin Fallavier, France). After centrifugation, cell pellet was suspended in DMEM and filtered through a 200 µm Nylon membrane and then a 50 µm Nylon membrane (Nytex, Dutscher Brumath, France). The SV cell sub-fractions isolated from adipose tissue or skeletal muscle were placed in 90% Fetal Calf Serum (FCS) and 10% dimethyl sulfoxide (1 to 2 million cells per mL) and frozen at -150°C.