Primary Bone Marrow Macrophage Isolation

Primary bone marrow macrophage (BMM) isolation was as previously described^{(18 (link))}. Bone marrow was extracted from long bones of 6–10 week old mice with αMEM (Sigma) in 10% FBS (Gibco) containing 100 IU/ml penicillin and streptomycin, and BMMs expanded in 1:10 dilution of CMG 14-12 cell supernatant (containing M-CSF, equivalent to 100ng/mL) for 4 days. For osteoclast differentiation, cells were cultured at the following densities: 9x10³ cells per well in 96-well tissue-culture plastic (TPP), 4.2x10⁴ in 48-well plates or 5x10⁵ in 6-well plates. 30 ng/ml GST-RANKL and CMG 14-12 supernatant supplying an equivalent 20 ng/ml M-CSF were added daily with media change until TRAP⁺ multinucleated cells became confluent. For TRAP staining, cells were fixed in 4% ultrapure formaldehyde (Polysciences) and 0.1% Triton X-100 in PBS for 5 min, and stained for TRAP according to the commercial kit protocol (Sigma).