Mineralization and Alizarin red S staining

Saos2 cells were cultured in osteoblastic differentiation medium containing 50 μg/ml ascorbic acid and 10 mM 3-glycerophosphate for up to 18 days. Differentiation medium was changed every other day. For the Sp1 over-expression experiment, cells were infected with Adenovirus-Sp1 or Adenovirus-β-gal for 48 hr and then cultured in differentiation medium for 9 and 18 days to determine the effects of Sp1 in early and late stage of differentiation and mineralization. For the MMA treatment, 100 nM MMA or equal volume of ethanol was added to the MMA treatment or control group, respectively. After 24 hr, the cells were cultured in the same differentiation condition as described above and fixed at day 14 for mineralization assay. Cells were also cultured in growth media without the supplements in parallel as an undifferentiated control for both experiments. Cell fixing and staining with Alizarin red-S and quantification of mineralization in these cells using cetylpyridinium chloride was performed as described [18].