Alkaline phosphatase (ALP) assay

ALP activity is the main biomarker of osteogenic differentiation. Alkaline Phosphatase Yellow (pNPP) Liquid Substrate (P7998-100ML, Sigma, USA) was used. The ALP assay was implemented on and days 3 and 5 after osteogenic induction. A mixture of the OM ingredient and the complete media in a 1:50 ratio was used for osteogenic induction media. The OM ingredient was prepared with 100 ml deionized water, 10.8 g β-glycerophosphate, 88.1 mg ascorbic acid, and 196.2 μg dexamethasone. Then, 3.0 × 10³ cells per well were seeded in a 96-well plate and incubated for 24 h at 37 °C. After that, they were washed with PBS and incubated with osteogenic induction media. At 3 and 5 days after osteogenic induction, ALP activity was measured. The cells were washed twice with a diluted assay buffer (10× Assay Buffer, ThermoFisher) with deionized water in a 1:10 ratio. After washing, 70 µl of the mixture of TritonX-100 (X100, Sigma, USA) and the diluted assay buffer in a ratio of 1:500 was added. The cells were placed in a 4 °C refrigerator for 10 min, and 50 ul diluted TritonX-100 was transferred to a new well plate. Then, 50 µl pNPP solution per well was added to the new well plate with diluted Triton X-100, incubated at 37 °C for 1 h and 15 min, and absorbance was measured by ELISA at 405 nm. Then, 5 µl of the remaining diluted TritonX-100 solution was transferred to a new well plate, and 200 µl of BCA solution (Pierce BCA protein assay reagent A, B, Thermo, USA) was added per well. After incubating at 37 °C for 30 min, the absorbance was measured at 570 nm with ELISA. To determine how much protein was present per well, absorbance data were calibrated using a BCA standard. The blank value was subtracted from the absorbance measured at 405 nm, and the result was divided by the amount of protein.