Alizarin Red Staining Protocol for Mineralization Nodule Assessment

After 14 days, the formation of mineralization nodules was assessed by alizarin red (Sigma) stain, following the manufacturer's instructions. Briefly, cells were fixed in 4% formaldehyde for 15 min, rinsed in PBS, and stained with 40 mM alizarin red staining reagent for 30 min. Cells were washed four times with distilled water, followed by an immediate 15-min rinse with PBS to reduce non-specific dying. The resulting samples were qualitatively analyzed and photographed under an inverted microscope^{80 (link)}. For quantitative analysis, a 4:1 ratio of 10% acetic acid (Dinâmica) and methanol (Dinâmica) was added to each well to dissolve calcified nodules. Then, the supernatant was read by a spectrophotometer at 405 nm^{82 (link)}. The test was performed in technical triplicate and three individual replicates, conducted on different days.