Alizarin red S and ALP staining

DPSCs were fixed with 4 % PFA for 1 h and washed with PBS. 2 % Alizarin red S solution was applied and DPSCs were placed in an incubator at 37 °C for 2 h. Mineralization was quantified by extracting the Alizarin red S stain with 100 mM cetylpyridinium chloride (Sigma-Aldrich) at room temperature for 2 h. Absorbance of the extracted Alizarin red S stain was measured at 570 nm. DPSCs were subjected to ALP staining using the ALP assay kit according to the manufacturer’s instructions. Alkaline phosphatase and Alizarin red S activities were determined by a colorimetric assay.