In Vitro Osteoclast Formation and Bone Resorption Assay

For osteoclast formation assays, BMMs seeded in 96-well plates at a density of 8×10³ cells/well in complete α-MEM containing 30 ng/ml M-CSF and 50 ng/ml RANKL were treated without or with various concentrations (5–20 μM) of Preg for 7 d. Medium containing M-CSF, RANKL, and Preg were changed every 2 d. At the end of 7 d, cells were fixed with 4% paraformaldehyde (PFA) for 20 min and stained for TRAP activity. TRAP stained cells were imaged under light microscopy and the number and size (in terms of area) of TRAP^+ve multinucleated osteoclasts with three or more nuclei were quantified using ImageJ software. For bone resorption assays, BMMs were cultured in six-well plates with 30 ng/ml M-CSF and 50 ng/ml RANKL for 3–4 d until small pre-osteoclast-like cells formed. Removed the cells with trypsin for 3 min and gently scraped them off and then centrifuge at 1,000 rpm for 5 min. Then equally reseed the cells onto hydroxyapatite-coated plates. Let the cells settle for 8 h, then treated with various concentrations (5, 10, and 20 μM) of Preg. After 4 d, adherent cells were incubated with 5% sodium hypochlorite solution for 15 min and then washed three times with PBS. Plates were air-dried and resorption pits visualized under light microscopy. The percentage of resorbed area relative to total well area for each experimental condition were measured using ImageJ software.