Osteoclastogenesis assays and TRAP staining

RAW264.7 cells were cultured in 96-well plates in DMEM supplemented with 10% FBS and 1% P/S at a density of 1×10⁴ cells/well. Cells were cultured for 6 days with RANKL (30 ng/ml) pre-incubated for 10 min with crosslinking anti-poly-histidine antibody (2.5 µg/ml; IC050P; R&D Systems, Inc., Minneapolis, MN, USA) to induce osteoclast formation (21,22), in the presence or absence of vehicle (ethanol; final concentration 0.1%), alendronate (0.1, 1, 10 or 100 µM), genistein (0.1, 1, 10 or 100 µM), or alendronate (0.1, 1, 10 or 100 µM) plus genistein (0.1, 1, 10 or 100 µM). After 6 days of culture, the cells were fixed and stained for TRAP, a specific marker of the osteoclast phenotype, using a leukocyte acid phosphatase kit. Briefly, cells were washed with PBS, fixed with 10% neutralized formalin-phosphate (pH 7.2) for 10 min, dried and stained with a leukocyte acid phosphatase kit (387A; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for staining of tartrate resistant acid phosphatase (TRAP) at room temperature for 90 min. TRAP-positive multinucleated cells (MNCs with at least three nuclei) were considered to be osteoclast-like cells, and the cells were counted using light microscopy (Olympus MTV-3). MNC scores are expressed as the mean ± standard deviation of six cultures with two replicate wells per data set using different dishes and cell preparation.