2.5. Alkaline Phosphatase (ALP) Activity Assay

Early stage bone differentiation indicator, or ALP activity, was examined using the ALP detection kit. MSCs at a concentration of 1 × 10⁴ cells/mL were seeded and cultured in the presence of test materials for 7 and 14 days in bone differentiation cell media. Bone differentiation culture media was prepared by adding 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone into the general cell culture media. Adhered cells were rinsed with PBS thrice and lysed with Triton X-100 for 5 min. The protein quantity was determined by the Bradford assay kit, and an equal amount of protein was compensated into samples. Samples were reacted with para-nitrophenyl phosphate (pNPP) and measured at 405 nm wavelength.