Measurements of alkaline phosphatase activity

After transfection with ING1b or HIF1α, C3H10T1/2 mesenchymal stem cells and MC3T3-E1 preosteoblasts were cultured in an osteogenic differentiation medium for 3, 5, 7, and 9 days. Cells were lysed using a commercial cell lysis solution according to the manufacturer's instructions. The alkaline phosphatase (ALP) activity assay was performed using a commercial Alkaline Phosphatase Detection Kit (Nanjing Jiancheng Bioengineering Institute). Cell lysates were incubated with ALP assay working solution (containing 1 mM pNPP). The absorbance of the samples was determined by using an ELISA plate reader at 405 nm (Fung et al., 2014).