Alizarin Red S (ARS) staining and quantification

To visualize the mineralization potential of ligament cells, ARS staining was performed, as described previously (18). ARS selectively binds to calcium, yielding a dark red color. Before ARS staining, OPLL cells (1x10⁵ cells/well) were cultured in a 12-well plate with osteogenic induction medium for 7 days, as aforementioned. The cultured cells were fixed with 95% ethanol for 30 min at room temperature, and then stained with 0.1% ARS (Sciencell Research Laboratories, Inc.) for 20 min at room temperature. Images of the calcified matrices were photographed using a conventional camera.

An ARS Staining Quantification Assay kit (Sciencell Research Laboratories, Inc.) was used for the quantification of matrix mineralization. Basically, the stain was eluted using 100 mM cetylpyridinium chloride for 1 h and spectrophotometric quantification was performed at 562 nm.