Alkaline phosphatase (ALP) assay

The MC3T3-E1 cells (2 × 10⁵) were plated in 24-well plates and cultured in the osteogenic media for 1 day, and then treated with or without rhBMP-2. The medium and rhBMP-2 were renewed every 3 days. After the treatment with rhBMP-2 at the required dose and time, the cells were washed twice with phosphate-buffered saline (PBS). Subsequently, lysis buffer 500 μL (10 mMTris-HCl (pH 7.5), 0.5 mM MgCl₂, 0.1% Triton-X) was added and the resulting mixture was sonicated on ice to lyse the cells. The protein concentrations were measured using the Bradford protein assay. The final concentration of the protein used in this work was 50 μg. The lysates were incubated with 200 μL ALP reaction buffer [25 mM Glycine (pH 9.4), 0.1% Triton X-100, 2 mM MgCl₂, and 5mMp-nitrophenyl phosphate] for 1 h at 37 °C. The reaction was quenched using 200μL of 1 M NaOH, and the ALP activity was measured from the optical absorbance at 405 nm using a microplate reader.