Osteo assay surface 24 well plate

RAW264.7 cells were cultured in Corning Osteo Assay Surface 24-well plates coated with calcium phosphate substrate at the density of 2 × 10⁴ cells/well. The medium was removed after 24 h incubation and inducing medium containing different dose of Au₂₅Sv₉ (0.4, 2 and 4 µM) was added. Culture medium was refreshed every two days. After about 6 days, cells were removed with a 10% sodium hypochlorite and rinsed for three times with water. The bone resorption pits were observed and photographed by light microscope. Image J software was use to quantified the percentage of resorbed bone surface area.

BMMs (1 × 10⁵ cells/cm²) were cultured in medium supplemented with 30 ng/ml M-CSF and 100 ng/ml receptor activator of nuclear factor kappa-Β ligand (RANKL). The cells were collected at 48 h to determine osteoclastic differentiation by measuring the messenger RNA (mRNA) expression of osteoclastic markers (Nfatc1, cathepsin K, Mcsfr and TRAP). As for TRAP staining, cells at 4 days were fixed at room temperature with 4% formaldehyde for 30 min. The cells were washed with PBS twice and then stained with a TRAP staining solution at 37°C for 30 min. TRAP-positive multinucleated cells with more than three nuclei were counted. As for the pit formation assay, BMMs were seeded in Osteo Assay Surface 24-well plates (Corning, NY, USA) at a density of 2.5 × 10⁴ cells/well and then cultured in medium supplemented with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for 4 days. The media was then removed from the wells, and 100 μL of a 10% bleach solution was added. The cells were incubated with the bleach solution at room temperature for 5 min. Then, the wells were washed twice with distilled water and dried at room temperature for 5 h. Individual pits or multiple pit clusters were observed by a microscope at 100× magnification. The ratio of the resorbed area to the total area was calculated using ImageJ software.