Type 2 collagenase from clostridium histolyticum

Osteoblast/osteocyte-enriched cells were obtained following a modification of a previously described method [Halleux et al., 2012 (link)]. Femurs were removed aseptically from 1 month old Dmp1-Cre^+/−;Rosa^Notch/WT and Rosa^Notch/WT control mice; the surrounding tissues were dissected, the proximal epiphyseal end was excised, and the bone marrow was removed by centrifugation. The distal epiphysis was excised, and femurs were digested for 20 min at 37°C with type II collagenase from Clostridium histolyticum (Worthington Biochemical Corp., Lakewood, NJ) pretreated with N-α-tosyl-L-lysyl-chloromethylketone hydrochloride) (Calbiochem, La Jolla, CA) and subsequently exposed to 5mM EDTA (Life Technologies, Grand Island, NY) for 20 min at 37°C. The resulting terminally differentiated osteoblast/osteocyte-enriched cortical femurs were cultured in DMEM supplemented with non-essential amino acids (both from Life Technologies), 100 μg/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO), and heat-inactivated 10% fetal bovine serum (FBS, Atlanta Biologicals, Inc., Atlanta, GA) for 4 days in a humidified 5% CO₂ incubator at 37°C.