α mem medium

The chondrogenic ATDC5 cells were purchased from Sigma and cultured in DMEM/Ham’s F12 medium supplemented with 2% FBS, 2 mM l-glutamine and 1% penicillin/streptomycin. In addition, primary osteoprogenitors (COB) were isolated from calvaria of 5-day-old wild-type neonates (C57BL/6 J) using Collagenase type II (50 mg/ml, Worthington, LS004176) and Dispase II (100 mg/ml, Roche, 10165859001) and were maintained in α-MEM medium (Gibco) containing 10% FBS (Gibco), 2 mM l-glutamine (Corning), 1% penicillin/streptomycin (Corning), and 1% nonessential amino acids (Corning). COBs were differentiated with ascorbic acid (200 uM, Sigma, A8960) and β-glycerophosphate (10 mM, Sigma, G9422). Finally, bone marrow cells were flushed from the femurs and tibias of 2-month-old mice (C57BL/6 J), and cultured in petri dishes in α-MEM medium with 10% FBS and 20 ng/ml of M-CSF (R&D systems) to obtain bone BM-OCP. After 12 h, nonadherent cells were replated into tissue culture dishes and cultured in the same medium for 3 days. BM-OCPs then differentiated into osteoclasts in the presence of RAMKL (20 ng/ml; R&D systems) and M-CSF (20 ng/ml; R&D systems) for 6 days.

The recovered fat tissues were transferred to 50 mL Falcon tubes and weighted. For enzymatical dissociation, an equal volume of 250 U/mL of collagenase type II (Ref C6885, Sigma, Saint-Quentin Fallavier, France) was added to the fat tissue and incubated at 37°C for 45 min under agitation as previously described (11 (link)). Cell suspensions were filtered using a sterile tea strainer and centrifuged at 300 × g for 10 min. The SVFs were collected, treated with erythrocyte lysis buffer (155 mmol/L NH₄Cl, 10 mmol/L KHCO₃, 0.11 mmol/L EDTA) and filtered successively through 100, 70, and 40 μm porous filters (Cell Strainer, BD Biosciences, le Pont-de-Claix, France). Cells were centrifuged at 300 × g for 5 min and resuspended in expansion medium (α-MEM medium supplemented with 100 U/mL penicillin/streptomycin, 2 mmol/mL glutamine, 10% fetal calf serum and 1 ng/mL bFGF) (R&D Systems, Abingdon, UK). Quantification of cells was performed using a hemocytometer and cells were plated at 4000 cells/cm² for 7 days. On day 7, cells were trypsinized, counted, and replated in expansion medium at the density of 2000 cells/cm² for another period of 7 days (end of passage 1).