Leukocyte acid phosphatase kit

Cells were seeded in 96-well plates (40,000 cells/mL), and test compounds were added 7–16 h later. In order to evaluate osteoclast differentiation, both TRAP activity and the number of TRAP-positive multinucleated cells were examined. TRAP activity in the cells was determined after 2–3 days by fixation with formaldehyde for 5 min (3.7% v/v) and washing with ethanol (95% v/v) for one min, followed by incubating the cells with 10–20 mM p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 10 mM sodium tartrate. The reaction was stopped with 0.1 M NaOH, and absorbance was measured at 410 nm. TRAP levels were corrected to cell number using crystal violet. Briefly, after fixation, cells were incubated for 15 min with crystal violet (0.5%) and washed thoroughly in tap water. After drying overnight, the dye was dissolved in sodium citrate, and absorbance was measured at 550 nm. It should be noted that the values of crystal violet staining, after treatment of cells with the various dietary compounds, did not differ by more than 20% from the value measured with RANKL alone. After 4 days, cells were fixed and stained for TRAP using a Leukocyte Acid Phosphatase kit (Sigma-Aldrich, St. Louis, MO, USA). The number of TRAP-positive multinucleated (>5 nucleus) cells was counted under a light microscope.

Preosteoclasts were generated as described above and incubated for 1 h with 10 μg ml⁻¹ of blocking antibodies against FcγRI (10.1, biolegend), FcγRII (AT10, abcam), FcγRIII (3G8, provided from the group of Falk Nimmerjahn) or isotype control (mouse IgG1, κ, biolegend), respectively. Subsequently, 100 μg ml⁻¹ of desialylated aggregated IgG was added for 6 h, followed by a complete medium exchange. After additional 18 h, osteoclast differentiation was evaluated by staining cells for TRAP using a Leukocyte Acid Phosphatase Kit (Sigma) according to the manufacturer’s instructions.