Alizarin Red S staining

BMSCs were fixed with 70% ethanol and then stained with 2% Alizarin Red S (MilliporeSigma). After washing in phosphate-buffered saline (PBS), the images of stained cells were visualized under a light microscope (Leica Microsystems, Germany). For quantification of mineralization, cells were incubated for one hour with 1 ml of cetylpyridinium chloride buffer to extract Alizarin Red S. Afterwards, the level of Alizarin Red S in total protein was determined by monitoring the absorbance at 562 nm via a microplate reader (Thermo Fisher Scientific). Alizarin Red S accumulation was expressed as μmol/μg protein.