5 bromo 4 chloro 3 indolyl phosphate bcip nitro blue tetrazolium alkaline phosphatase color development kit

The cyclopamine treatment processes were the same as those in the cell proliferation assay. The MG63 cells were seeded onto the different titanium surfaces in 12-well plates with a concentration of 5×10⁴/well for 7 days. Then, the cells were cleaned gently with PBS and fixed with 4% polyoxymethylene for 15 minutes. ALP staining was performed with a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) Alkaline Phosphatase Color Development Kit (Beyotime). The ALP activity was assessed using a quantitative ALP assay kit (Beyotime) according to the guidelines of the manufacturer. Briefly, the MG63 cells were cleaned twice with PBS and, then, lysed with ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Beyotime). The aliquots of supernatants were added with substrates and p-nitrophenol and incubated for 15 minutes at 37°C. The absorbance of p-nitrophenol formed was detected using a microplate reader at a wave length of 405 nm. The amount of intracellular total protein was detected using the BCA Protein Assay Kit (Thermo Fisher Scientific) and the ALP activity was normalized to the total protein content reported as units per milligram of protein.