4.6. Alkaline Phosphatase (ALP) Activity Assay

The ALP activity of the BMSCs and DPSCs was determined by measuring the rate of conversion of p-nitrophenyl phosphate (p-NPP) to p-nitrophenol (p-NP) at a pH of 10.2 and temperature of 37 °C. The cells were seeded in 24-well plates at a density of 1.0 × 10⁴ cells/ml in normal culture and osteogenic medium. After incubation for 1, 2, 3, 4, 5, and 6 days, the cells were washed with PBS and lysed with 300 μl CelLyticTMP Cell Lysis Reagent (Sigma–Aldrich Co., St. Louis, MO, USA). The cell lysate was mixed and incubated with ALP assay reagent at 37 °C for 30 min and the absorbance of the resulting ALP activity was quantified by calculating the optical density (OD) values at 405 nm. The total protein content of each cell lysate was measured using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). For each cell type, the ALP enzymatic activity was expressed as micromoles of p-nitrophenol produced per milligram of protein per minute.