Induction of Osteoblast Mineralization and Alizarin Red Staining

Alizarin red dye can stain the mineralization nodules produced by osteoblasts, which could be used to identify isolated primary cells or to evaluate the effect of C3G on the mineralization ability of osteoblasts. The cell density was adjusted to 7 × 10⁵/mL, then inoculated into a Petri dish having a diameter of 60 mm, and the inoculum amount per dish was 1 mL. In the study for the identification of osteoblasts, the cells were cultured for 14 days, and then the medium in the dish was changed to a complete medium containing 10 mmol/L of β-glycerophosphate. The treatments were continued for 14 days, and the sample was stained according to the method of using the alizarin red dye. In an assay for evaluating the mineralization capacity of C3G on osteoblasts, cells were seeded in 12-well plates. The density of the seeded cells was 1 × 10⁴/mL, and the inoculum amount was 1 mL per well. Primary osteoblasts were cultured for 7 days in a complete medium containing various concentrations of C3G, changing every 2 days because of the poor stability of C3G. After 7 days, the medium in the wells was removed, and after the cells were fixed as necessary, they would be stained with alizarin red dye. The appearance of calcified nodules was examined under an inverted microscope and photographed and stored.