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After hPDLSCs were cocultured using OM and 1,25(OH)2VitD3 for 7 days, calcification of hPDLSCs was observed using alizarin red S staining solution (G1452; Solarbio). After the culture medium was removed, the cells were fixed in 4% paraformaldehyde for 15 min. Then, 1% alizarin red S solution was added to evenly cover the cells for staining for 5 min and the mineralized nodules were observed under microscope.
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