Alizarin Red S Staining

Alizarin Red S Staining was performing on day 28 of osteoblast differentiation. Briefly, hBMSCs were washed with PBS and fixed with 70% ice-cold ethanol for 30 min at room temperature. Then, cells were stained with 2% Alizarin red S staining kit (Sigma, St. Louis, Missouri) for 30 min at room temperature. Subsequently, cells were washed with water and observed under an inverted microscope. About 10% acetic acid was used to extract the alizarin red S in cell culture, then 10% ammonium hydroxide was used to neutralize the acid. The absorbance of cell culture extractions and alizarin red S standard at 550 nm were read with a plate reader.