Bone resorption pit assays.

The bone resorption activity of osteoclasts was examined using 96-well Corning Osteo Assay Surface Plates (Sigma-Aldrich). Mouse OCPs were plated at a seeding density of 1 × 10⁴ per well and incubated with M-CSF–containing CM and RANKL (50 ng/ml) for 10 days, with exchange of fresh M-CSF–containing CM and RANKL every 2 days. After removing cells with 10% bleach solution, plates were stained with 1% toluidine blue solution to visualize the formation of pits. The resorbed area was analyzed using OsteoMeasure software (OsteoMetrics).