Cell culture and osteoclast formation

The RAW 264.7 (RAW) murine macrophage cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA). RAW cells were cultured and maintained in complete DMEM containing 10% heat-inactivated FBS at 37°C, 5% CO₂, and 90% relative humidity in T75 flasks. To generate osteoclasts, confluent RAW cells were subcultured at a density of 10,000 cells/cm² into 12-well or 24-well tissue culture plates in AMEM supplemented with 10% FBS and 25–100 ng/ml RANKL. The medium was replaced every 2 d with cell fusion typically occurring within 2 d from initial seeding and large multinucleated osteoclasts forming on days 3 and 4.