Alkaline phosphatase (ALP) activity assay

To evaluate osteoblastic differentiation, alkaline phosphatase (ALP) activity was measured at 3, 7, and 14 days. ALP activity was performed according to the pNPP method [24]. Cells were plated in 24-well plates at a density of 2 × 10⁴ cells/well. Following treatment with rhBMP-2 and LLL irradiation, the cells were washed twice with PBS and scraped into 10 mM Tris–HCl containing 2 mM MgCl₂ and 0.05% Triton X-100 (pH 8.2), placed on ice, and sonicated to lyse the cells. Protein concentrations in the lysates were determined by using the Bradford protein assay. The cell lysates were mixed with an aliquot of assay buffer containing 10 mM p-nitrophenyl phosphate in 0.1 M sodium carbonate (pH 10.2) supplemented with 1 mM MgCl₂ followed by incubation at 37 °C for 30 min. After incubating at 37 °C for 30 min, the reaction was stopped by adding 1 M NaOH and the amount of p-nitrophenol released was estimated by measuring the absorbance at 405 nm. Relative ALP activity is defined as millimoles of pNPP hydrolyzed per min per mg of total protein.