Osteoclast Bone Resorption Assay

WT- or Parkin^−/−-derived macrophages (1 × 10⁵ cells) were cultured on dentine slices for 24 h in α-MEM medium containing 30 ng/ml M-CSF and 10 ng/ml RANKL and further incubated for 7 days. The medium was then removed, and 1 M NH₄OH was added to the wells for 30 min. Adherent cells were removed from the dentine slices by ultrasonication; the resorption areas were visualized by staining with 1% toluidine blue. The resorbed pit area on the dentine was then photographed using an image analysis system (NIS-Elements Imaging Software) linked to a light microscope (Nikon). For inflammatory IL-1β-induced OC bone resorption, WT or Parkin^−/− OCPs primed with RANKL on a bone slice for 24 h were further incubated for 7 days in the presence of 10 ng/ml IL-1β, and the resorbed pit area sizes were evaluated, as described above. Resorption pit areas were quantified by ImageJ software.