Alkaline Phosphatase Activity Assay

Alkaline phosphatase (ALP) enzymatic assay was measured using a colorimetric method based on the conversion of p-nitrophenyl phosphate into p-nitrophenol in the presence of ALP (ABCAM). hFOB spheroid after 3, 7, and 14 days of culture were washed gently with PBS, followed by incubated with 100 μl of ALP assay buffer for lysate the spheroids. Then, the lysates (50 µl) were added in a 96-well-plate adjusted to 80 µl with assay buffer, and then 50 µl of 5 mM of p-nitrophenol (p-NP) solution were added. The well plate was incubated protected from light at 25°C for 60 min, and the reaction was stopped by the addition of 20 μl stop solution. The colorimetric reaction was presented as absorbance and was read at 405 nm with an ELISA plate reader (ChroMate, AWARENESS Technology).