Human csf1

For mouse primary cell culture, bone marrow cells isolated from mice were cultured as described previously18 (link)19 (link). Briefly, Bone marrow cells were isolated from flushing the femurs and tibias of 6- to 8-week-old C57BL/6 mice. To generate BMMs, the cells were cultured in α-MEM with 10% FBS containing 20 ng/ml M-CSF. To generate osteoclasts, the BMMs were seeded into 96-well plates and incubated with M-SCF (20 ng/ml) 2–3 days before stimulation with RANKL (30 ng/ml). After 6 or 4 days, cells were fixed and stained for Tartrate-resistant acid phosphatase (TRAP) activity (Sigma). TRAP positive multinucleated cells with more than 5 nuclei were counted as osteoclasts. For human osteoclastogenesis assay, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor by Ficoll gradient centrifugation (provide by Shanghai Blood Center). The culture medium consisting of alpha minimal essential medium (α-MEM) supplemented with 10% foetal bovine serum (FBS). For osteoclastogenesis, 5 × 10⁵ PBMCs were seeded in a 96-well plate with 20 ng/ml human CSF1 (Sino Biological Inc, 11792-H08Y). After 36 hours, cells were stimulated with 50 ng/mL human RANKL (R&D, 6449-TEC) and 20 ng/mL human CSF1 for 8–9 days. Medium was changed every two day. Osteoclasts were fixed and stained using the TRAP staining kit (Sigma, 387A-1KT).

Human PBMCs were isolated from healthy donors by Ficoll gradient centrifugation. Written informed consent was obtained from all donors. PBMCs were cultured in ɑ-MEM with 10% FBS (Sigma), 1% GlutaMAX Supplement (Thermo Fisher) and 30 ng ml⁻¹ human CSF-1 (Sino Biological, 11792-H08Y) and incubated in a humidified atmosphere at 37 °C and 5% CO₂. For osteoclastogenesis, 5 × 10⁵ PBMCs were seeded in a 12-well plate, after 48 h, cells were stimulated with 50 ng ml⁻¹ human RANKL (R&D, 6449–TEC-010) and 30 ng ml⁻¹ human CSF-1 for 10–14 d. Medium was changed every 2 d. Osteoclasts were fixed and stained using the TRAP staining kit (Sigma-Aldrich, 387A-1KT).