Alizarin Red S Staining Quantification Assay

Alizarin Red S (ARS), an anthraquinone dye, has been widely used to evaluate calcium deposits in cell culture. The ARS staining is quite versatile because the dye can be extracted from the stained monolayer of cells and readily assayed.

ScienCell’s ARS Staining Quantification Assay (ARed-Q) provides a sensitive tool for the recovery and semi-quantification of ARS in a stained monolayer of cells. Mineralization is assessed by extraction of calcified mineral at low pH, neutralization with ammonium hydroxide, and colorimetric detection at 405 nm in a 96-well format. This assay is more sensitive than the cetylpyridinium chloride (CPC) extraction method, improving the detection of weakly mineralizing monolayers [1]. It provides a wider linear range: destained ARS dye ranging from 30 µM to 4 mM shows a linear relationship with the absorbance at 405 nm, making dilutions of samples prior to measurement unnecessary.

We have applied this quantification assay to the osteogenesis induction of human mesenchymal stem cells (hMSCs). Cells were cultured in different osteogenic differentiation medium for 18 days, fixed for ARS staining and quantified for mineral deposit using the kit. There is significant difference between the untreated control, the induced samples, and the different induction conditions (Figure 2). ScienCell’s ARed-Q assay can be applied for mesenchymal stem cell osteogenic differentiation, tumor characterization, and osteognic compound screening.