Quantifying Osteogenic Differentiation via ALP Activity

After 7 days of osteogenic induction, alkaline phosphatase (ALP) staining was performed using a BCIP/NBT ALP color development kit (Beyotime, C3206, China) following manufacturer’s instruction. Briefly, after removing the culture medium, the cells were washed three times with PBS and fixed with 4% polyoxymethylene for 15 min. Then, the cells were immersed by BCIP/NBT staining working solution and incubated at room temperature for 30 min in dark. The color reaction was terminated by distilled water. ALP quantification was performed with an alkaline phosphatase assay kit (Beyotime, P0321M, China) according to the manufacturer’s protocol. The optical density (OD) was evaluated by measuring the absorbance at 405 nm spectrophotometrically. The total protein content was measured at OD_562nm, and the concentration was calculated according to the standard BSA curve using the enhanced BCA protein assay (Beyotime, P0010) [33 (link)]. ALP activity was represented by the OD_405nm value after normalization to the total cellular protein.