Protocol(for reference only)

1. Inoculated cells: the cells of logarithmic growth stage were inoculated into the coated culture vessel according to the cell density of 2×104cells/cm2, and cultured at 37℃ and 5% CO2 until the fusion degree was 60-70%. The supernant was discarded and added into the osteogenic induction differentiation medium.

2. Induction of cell differentiation: Replace osteogenic induction medium every 2-3 days, culture at 37℃, 5%CO2 culture environment, and pay attention to the changes in cell morphology (generally about 14 to 28 days). According to the precipitation of calcium salts and the formation of calcium nodules, the time of termination of cell induction was determined and staining identification was performed.

3. Cell fixation: Remove the culture medium and wash it once with an appropriate amount of 1×PBS, then cover the bottom surface of the culture vessel with an appropriate amount of 4% neutral formaldehyde solution after discarding, fix it at room temperature for 30-60 min, discard the fixing solution and wash it twice with 1×PBS.

4. Alizarin red staining: Add an appropriate amount of alizarin red dye solution for 3~5min, absorb alizarin red dye solution, wash twice with 1×PBS, and add an appropriate amount of 1×PBS to avoid cell drying.

5. Induction evaluation: The effect of osteogenic staining was observed under a microscope, and image collection and induction evaluation were carried out. When induction is successful, the calcium nodules will appear red or orange-red after binding with alizarin red dye.

6. Semi-quantitative analysis: After the microscopic observation, the supernatant was abandoned, cetylpyridine chloride solution was added, and the mineralized nodules (alizarin red) were dissolved by incubation at room temperature for 15-60 min. Then the OD value of the supernatant was detected at 562 nm.