Alizarin red staining

Matrix mineralization, which is mediated by the well-differentiated osteoblasts, was measured by Alizarin red staining. Cells were washed with PBS and then directly fixed with ethanol at − 20 °C for at least 60 min. After fixation, cells were washed by using tap water three times and incubated with Alizarin red solution (0.5 % w/v, pH = 4.0) for 30 min at room temperature. Stained matrix (red) can be detected by microscope after washing with tap water at this stage. The bound staining was resolved with 10 %w/v cetylpyridinium chloride solution, and the quantification of alizarin staining was determined by measuring the absorbance (λ = 562 nm) using a plate reader (Ehnert et al., 2015[12]).