2.8. Alizarin Red staining and calcium assay

Mineral deposition was confirmed by Alizarin Red staining and calcium assay according to the manufacturer's instructions. Briefly, the cells were washed thrice with PBS and fixed with glutaraldehyde and then were incubated with Alizarin Red S (Beyotime Institute of Biotechnology) for 10 minutes at room temperature. After washing with PBS, the images of calcium nodules were taken by microscope. Alizarin Red dye was quantified by spectrophotometry and normalized to protein level. After washing with PBS for three times, the samples were decalcified with 0.6 mmol/L HCl at 4°C for 24 hours. Calcium was detected with Arsenazo III calcium measurement kit (StanBio Laboratories Inc) according to the manufacturer's protocol. Protein content was measured using BCA protein assay. The amount of calcium was normalized to total protein and expressed as fold change compared with control.