4.4. In Vitro Osteoclastogenesis Assays

RAW264.7 cells were plated in 24-well plates (1 × 10⁴ cells/well) and cultured in DMEM treated with DFO (0, 10, 20, 50, and 100 µM) for 2 h. Next, DMEM was changed with Minimum Essential Medium (MEM) Alpha (Invitrogen) containing 10% FBS and 50 ng/mL of receptor activator of nuclear factor-κB ligand (RANKL) (PeproTech Inc., Rocky Hill, NJ, USA), for 3 days at 37 °C in a CO₂ incubator.