Spectramax 50s

The enzymatic activity of ALP was determined using a TRACP & ALP Assay Kit (TaKaRa Bio, Shiga, Japan) in accordance with the manufacturer’s protocols. Briefly, the cells were washed with saline and solubilized with 1% NP-40. The cell extracts were incubated in solution containing p-nitrophenyl phosphate at 37 °C. The reaction was stopped by the addition of 0.5 N NaOH, and the absorbance at 405 nm was measured with a microplate reader SpectraMax 50S (Molecular Devices Japan, Tokyo, Japan). A standard curve was constructed using calf intestinal ALP (TaKaRa Bio), and the ALP activity in the samples was calculated. One unit (U) of ALP activity represents the hydrolysis of 1 μmol p-nitrophenyl phosphate per minute at 37 °C.

The mineralization of MC3T3-E1 cells was determined by staining with Alizarin Red S, which binds to calcium phosphate and produces red staining. The cells were washed with phosphate-buffered saline and fixed with 10% neutral-buffered formalin solution for 15 min at room temperature. The cells were then incubated with Alizarin Red S solution (PG Research, Tokyo, Japan) for 30 min at room temperature. After washing with distilled water, 5% formic acid was added to the plates, and the absorbance at 415 nm was measured with a microplate reader SpectraMax 50S (Molecular Devices Japan).