Isolation and Culture of Murine Bone Marrow Macrophages

WT or PIP5k1β^−/− BMMs were obtained as described previously (Qin et al., 2012 (link); Li et al., 2013 (link)). In brief, bone marrow cells extracted from the femurs and tibiae of 9–10-week-old WT and littermate PIP5k1β^−/− mice were cultured in α-MEM medium containing 15 ng/ml M-CSF (416-ML; R&D) in a T-75 cm² flask for proliferation for 2–3 days until reaching 90% confluence. The cells were washed with phosphate buffer solution (PBS) three times and trypsinized for 30 min. Adherent cells were classified as BMMs. WT and PIP5k1β^−/− BMMs were plated on 96-well plates at a density of 8 × 10³ cells/well in triplicate and incubated with α-MEM medium containing 20 ng/ml M-CSF in a humidified incubator containing 5% CO₂ at 37°C for 2 days. The cells were then cultured with α-MEM medium with M-CSF (20 ng/ml), RANKL (75 ng /ml; 315-11; Peprotech), and indicated inhibitors for indicated times. The medium was changed every other day. The inhibitors for p38/MAPK (SB203580), MAPK/ERK (U0126-EtOH), and JNK (SP600125) were purchased from Selleckchem. For osteoblast differentiation, BMSCs were gained from 9–10-week-old mice and expanded as previously described, induced by culturing cells in osteogenic medium (DMEM containing 1 M β-glycerophosphate, 50 mM ascorbic acid, and 1 mM methylisobutylxanthine) for indicated times, followed by subsequent experiments.