Isolation and Culture of Mouse Bone Marrow-Derived Macrophages and Osteoclasts

We cultured primary bone marrow cells isolated from C57/BL6 mice as described40 (link)41 (link). Briefly, Bone marrow cells were isolated from 8-week-old C57/BL6 mice by flushing femurs and tibias with α-MEM and cultured in α-MEM with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and M-CSF (30 ng/ml) overnight. Non-adherent cells were collected and further cultured in the presence of M-CSF (30 ng/ml) for 3 days. Floating cells were discarded and adherent cells were used as bone marrow-derived macrophages (BMMs). Preparation of mouse osteoclasts was carried out as described previously42 (link)43 (link). In brief, BMMs (4 × 10⁴ cells/well) were seeded on a 0.2% collagen-gel coated 12-well plate and induced by RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 6 days. Then osteoclasts were recovered by treatment with 0.2% collagenase, suspended in a-MEM containing 10% FBS, and used for osteoclast function assays.
Rat bone marrow mesenchymal stem cells (BMSCs) were isolated from 4-week-old Sprague–Dawley rats (male or female 80–100 g) and expanded in accordance with published techniques44 (link)45 (link)46 (link). The cells were maintained in expansion medium consisting of Dulbecco’s modified Eagle’s medium (DMEM)/F12, 10% FBS.