4.8. Alkaline Phosphatase (ALP) Staining and Alizarin Red S (ARS) Staining

Cells were cultured in 12-well plates supplied with osteo-/odontogenic differentiation induction medium. In different groups, EGCG was added into the medium at concentrations of 0, 0.1, 1, or 10 μM. The medium was renewed every three days. For ALP staining, after seven days of osteo-/odontogenic induction, cells from each group were fixed with 4% PFA. Then, cells were stained with CIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime). The quantitation of ALP activity was detected using an Alkaline Phosphatase Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The quantitative values were measured at 520 nm absorbance. The values of ALP activity were calculated in U/gprot.

For ARS staining, cells were stained with 1% Alizarin Red S after 14 days of osteo-/odontogenic induction. For semi-quantification of ARS staining, 10% (w/v) cetylpyridinium chloride was added to the plates to destain the samples. The absorbance was determined at 562 nm using a microplate reader.