Alizarin Red staining and mineralization assay

Cells were washed twice with cold PBS and fixed with 4% paraformaldehyde for 10 min. They were then stained with 50 mM Alizarin Red S (pH 4.2) for 10 min at room temperature, and images were obtained. In order to determine the extent of calcium deposition, the mineralization of calcium nodules was quantified. After staining, the cells were washed three times with PBS. Hexadecylpyridinium chloride monohydrate (10%) was added and incubated for 20 min at room temperature. The absorbance of the supernatant was measured at 562 nm in triplicate using a Multiskan EX plate reader (Power Wave XS₂; Thermo Fisher Scientific, Inc.). Finally, cells were washed in PBS and lysed with RIPA buffer. and protein content quantified by BCA and calcium levels were normalized to the total protein content.