Quantitative Bone Resorption Assay

Bone resorption activity was determined using bone resorption assay kit (CosMo Bio, Tokyo, Japan), as described previously [45 (link)]. Briefly, BMMs were cultured on a calcium phosphate-coated 48-well plate (2.5 × 10⁴ cells/well) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with different concentrations of PSTP-3,5-Me (0, 3, and 10 µM) for six days. The fluorescence intensity of the culture supernatant was analyzed using a fluorescence plate reader, SpectraMax i3x (Molecular Devices, San Jose, CA, USA) with 485 nm and 535 nm wavelengths. The pit area was calculated using Image J software.