Murine bone marrow macrophages were isolated from bone marrow flushed tibiae and femurs of WT and Rspo3+/- mice at 6–8 wk-old as described previously (Chen et al., 2019 (link)). Briefly, cells were cultured in complete α-MEM with 30 ng/ml macrophage colony-stimulating factor M-CSF (R&D system, Minneapolis, MN, USA) in suspension culture dish to which stromal cells and lymphoid cells cannot adhere, at 37 °C for 2–3 days. For osteoclast generation, cells were cultured in 30 ng/ml M-CSF and 10 ng/ml RANKL (R&D systems, Minneapolis, MN, USA). For co-culture experiments, mouse calvarial osteoblasts were isolated from newborn WT and Rspo3+/- as previously reported (Movérare-Skrtic et al., 2014 (link); Chen et al., 2019 (link)) and seeded in 96-well plates (2.000 cells/well) in complete osteogenic α-MEM containing 100 nM Vitamin D3 and 1 µM prostaglandin E2 (Enzo Life Science, Farmingdale, NY, USA). After 3 days, 10,000 BMM from WT and Rspo3+/- mice at 6–8 week-old mice were added per well and cocultured for 9 days in complete osteogenic α-MEM. Tartrate-resistant acid phosphatase (TRAP) staining was performed to evaluate the number of osteoclasts according to the manufacture’s protocol (Sigma-Aldrich, St. Louis, MO, USA).
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