Osteoclast Precursor Isolation Protocol

Osteoclast precursors were isolated as previously described (18 (link)). Briefly, femurs and tibia were harvested after mice were euthanized by CO₂ asphyxiation. Each bone was placed in a 0.7 ml microcentrifuge tube that was pierced with a 22 G needle at the bottom. The 0.7 ml tube was placed inside a 1.5 ml microcentrifuge tube and spun for 30 seconds at 16,000 g. The bone marrow cells were resuspended and maintained in α-minimum essential medium (α-MEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), penicillin-streptomycin-glutamine (Invitrogen), recombinant murine M-CSF (eBioscience Inc, San Diego, CA) at 20 ng/ml and recombinant murine GST-RANKL (the expression system for GST-RANKL was a kind gift from Prof. Steven Teitelbaum, Washington University in St. Louis) at 50 ng/ml. Treatment with Versene (Gibco) for 10 minutes was used to harvest cells.