For Alizarin Red staining, after washing in ddH2O three times formaldehyde-fixed cells were stained with 2% w/v Alizarin Red S solution (pH 4.1) (Sigma-Aldrich) for 20 minutes, followed by further ddH2O washing twice. Then 10% cetylpyridinium chloride (CPC; Sigma-Aldrich) was used to extract the dye by incubating the stained cells at 37 °C for 20 minutes. The absorbance at 550 nm was measured for quantification.
For qualitative detection of mouse matrix gla protein (MGP) secreted from mMSCs in condition medium, mMSCs were cultured in 6-cm culture dishes. Fresh induction medium was added into culture dishes 3 days before being collected for the assay. MGP assay was performed by MGP ELISA kit according to the manufacturer’s instruction (MyBioSource, San Diego, CA, USA) and the amount of MGP secretion was normalized by the cell number of the control group.
For qualitative detection of mouse matrix gla protein (MGP) secreted from mMSCs in condition medium, mMSCs were cultured in 6-cm culture dishes. Fresh induction medium was added into culture dishes 3 days before being collected for the assay. MGP assay was performed by MGP ELISA kit according to the manufacturer’s instruction (MyBioSource, San Diego, CA, USA) and the amount of MGP secretion was normalized by the cell number of the control group.