Nbt bcip alp staining kits

For cell osteogenic induction, the culture medium was changed by osteogenic differentiation medium (OS, growth medium supplemented with 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid). Alkaline phosphatase (ALP) activity and Alizarin Red Staining (ARS) were performed to evaluate the osteogenic potential. ALP activity assay and NBT/BCIP ALP staining kits (Beyotime Biotechnology, China) were used to analyze the ALP activity according to the manufacturer's instructions on day 7. For ARS staining, 10% ARS solution (Sigma-Aldrich) was used to staining cells on day 21 for 5 min and washed by water. Then red mineral deposition that stained by ARS quantitation was applied by measuring the absorbance at 562 nm after mineral nodes were desorbed by 10% (w/v) cetylpyridinium chloride (CPC, Sigma-Aldrich).

To induce osteogenic/dentinogenic differentiation in cells, the culture medium was replaced with osteogenic medium (OM) every 3 days. The cells were then cultured in OM medium for 7 and 14 days, and the osteogenic/dentinogenic potential was evaluated using ALP activity assay and NBT/BCIP ALP staining kits (Beyotime Biotechnology, China). For ARS staining, cells were stained with a 10 % ARS solution (Cyagen Biosciences Inc, China) for 14 and 21 days, washed with dd H2O, and mineral deposition was quantified using a 10 % (w/v) cetylpyridinium chloride solution (Sigma-Aldrich), and the staining intensity was quantified by measuring the absorbance at 562 nm in a microplate reader (Bio-Tek). All experiments were conducted in triplicate.