Sodium acetate trihydrate

RAW264.7 cells were seeded in 96-well plates containing α-MEM supplemented with 100 ng/mL RANKL at a density of 2 × 10³ cells/well. The cells were treated with MKE for five days. TRAP staining was performed using a TRAP staining reagent containing 5 mg naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO, USA), 500 μL of N,N-dimethylformamide (Wako), and 30 mg Fast red violet LB salt (Sigma-Aldrich) in 50 mL TRAP staining buffer. The TRAP staining buffer was prepared using 0.11% citric acid (Sigma-Aldrich), 14 mmol of sodium acetate trihydrate (Wako), and 10 mmol of sodium (+) tartrate dihydrate (Wako) in distilled water. Cells were fixed with 4% paraformaldehyde, washed with PBS, and 50 μL/well of TRAP staining reagent was added to stain the cells. TRAP-positive cells were dark red and cells containing three or more nuclei were classified as osteoclasts. TRAP activity was measured spectrophotometrically in 50 mM citrate buffer containing 10 mM sodium tartrate and 6 mM p-nitrophenyl phosphate (Sigma-Aldrich). Briefly, 100 μL of buffer was added to each well and reacted with 85 μL of 0.1 N NaOH after 20 min. The absorbance of the supernatant was measured at 405 nm using a microplate reader (Tecan).