Alizarin Red S Quantification of Osteogenic Differentiation

Calcium deposition on day 14 of osteogenic differentiation was measured by Alizarin red S staining as described in Dubon et al. [19 (link)]. Cells were fixed with PBS-buffered 3.7% formaldehyde for 30 min at RT, rinsed with distilled water, and stained with 2% (w/v) Alizarin red S (Sigma Aldrich) dissolved in distilled water (pH 4.2, adjusted with 10% NH₄OH, Sigma Aldrich) for 20 min. Stained cells were washed with distilled water and an image of the mineralization was taken. Then, the dye was eluted with 10% (w/v) cetylpyridinium chloride monohydrate (Sigma Aldrich) in 10 mM sodium phosphate (pH 7.0; Sigma Aldrich) for 1 h at RT, and the absorbance of the eluent was measured at 570 nm using the Multiskan™ GO microplate reader.