7. Alizarin red S staining

Mineralized nodules from differentiated cells were visualized through alizarin red S staining to evaluate the mineralization-inducing potentials of each specimen. Cells were seeded at a density of 2×10⁴ cells per well onto a 48-well plate and were cultured for 14 days with each specimen. Then, mineralization of the cells was evaluated through alizarin red S staining (Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells were fixed with 4% formalin for 1 hour at 4℃, washed three times with deionized water, and then stained with 40 mmol/L of alizarin red solution (pH=4.2). The stained cell plate was washed with deionized water, and the stained image was obtained with the scanner. To remove the stain, the samples were treated with 10% cetylpyridinium chloride solution (pH=7.0) for 15 minutes, and absorbance was measured at a wavelength of 540 nm with a standard solution for the quantitative assessment.