ALP staining and activity

MC3T3-E1 cells were cultured overnight in six-well plates at 3 × 10⁵ cells/well. Two days after transfection, the cells were treated with STEMPRO for 7 days, then ALP staining and activity assays were conduced. Subsequently, the cells were fixed with 95% ethanol (v/v) and then incubated with a substrate solution from an ALP staining kit (Beyotime^® Institute of Biotechnology, Shanghai, China) in the dark, according to the manufacturer’s protocol. For ALP activity assays, after incubation, the treated cells were washed twice with PBS, and 200 µl of lysis buffer was added to the cell layer and kept on ice for 5 min. The cell lysate was sonicated for 1 min and centrifuged at 1000 × g at 4 °C for 10 min. ALP activity was assayed by a spectrophotometric method using a LabAssay™ ALP kit. The absorbance at 405 nm of each well was measured with a microplate reader according to the manufacturer’s instruction⁴⁰.