Alizarin red s kit

After 10 days of culture, cells were collected and stained. For alizarin red staining and quantitative analysis, the cells were rinsed with PBS, fixed in 4% paraformaldehyde for 15 min, washed with ddH₂O three times, and then stained with Alizarin Red S Kit (Solarbio, China) for mineralized nodules. The cells were washed again before taking photos. After that, 10% cetylpyridinium chloride was used to dissolve the nodules, and the OD (optical density) was measured at 562 nm. For ALP staining and activity analysis, cells were rinsed with PBS and treated with 1% Triton X-100. The ALP activity of the cell lysates was tested with an ALP Activity Assay Kit (Beyotime, China). The protein concentrations were examined using an Enhanced BCA Protein Assay Kit (Beyotime) following the manufacturer’s instructions. For alcian blue staining and analysis, cells were also fixed in 4% paraformaldehyde for 10 min and then washed and stained with an Alcian Blue Staining Kit (ScienceCell, USA). After staining, the cells were washed with diH₂O 3 times, and 6 mol/L guanidine hydrochloride was added and incubated for 2 h at room temperature. The OD was measured at 630 nm, and the cells were observed and captured under an inverted microscope (Olympus IX73, Japan).