Alizarin Red Staining of Calcific Deposition

MDPC-23 cells were inoculated at the number of 1.25 × 10⁴/well and cultured with MM in 12-well plates until day four. At day four, IM described above in cell culture were added to culture media. Calcific deposition of cells was visualized using alizarin red staining by day eight. Briefly, cell monolayer was rinsed once by PBS and fixed in 10% formalin neutral buffer solution (060-01667, Wako) for 20 min at room temperature. Thereafter, cells were washed once using distilled water and stained by alizarin red s solution (1%, pH 4.1, 011-01192, Wako) for about five minutes at room temperature. Staining solution was discarded and cell monolayer was washed by distilled water for 2 h. The stained mineralized nodules were photographed by an inverted digital camera (Canon) and quantified using cetylpyridinium chloride (CPC). The detailed quantification method was described elsewhere [18 (link)].