Alizarin red s

To assess osteogenic differentiation, hBMSCs cells were transferred into 48-well plates at confluent cell density (10,000 cells/well). On the second day after plating, the culture medium was replaced with osteogenic differentiation medium (StemPro Osteogenesis Differentiation kit, Gibco). The medium was replaced with fresh medium every 3 d. The cells were cultured for 21 d and their osteogenic differentiation was analyzed by staining with Alizarin red S (Sigma Aldrich). The cells were washed with PBS and then fixed in 4% paraformaldehyde (Thermo Fisher Scientific) for 10 min. They were then washed with PBS twice and stained with Alizarin red S for 15 min, followed by washing with PBS another three times. The cells stained with Alizarin red S were imaged using a Leica DMi1 Inverted Microscope. To quantitate Alizarin red Staining, the stained cells were rinsed with PBS and extracted with 10% (w/v) cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0) for 10 min at room temperature. Alizarin red in the extract was quantitated by optical density measurement at 562 nm.

Calcification in the mouse aorta was observed using Von Kossa staining kit (ab150687, Abcam, UK). Briefly, aortic tissue sections were incubated with 5% silver nitrate solution for 30 min under a UV lamp and then treated with 5% sodium thiosulfate. Subsequently, the sections were counterstained using Nuclear Fast Red solution. Ca deposits were stained brown-black and observed under an inverted microscope (Leica, Germany).
Calcification of the MOVAS cells was observed using the Alizarin Red S staining method. Briefly, the cells were fixed in 4% formaldehyde for 5 min at 25°C, exposed to 2% Alizarin Red S (A5533, Sigma) for 30 min, and washed with 0.2% acetic acid. Calcified nodules were stained red and observed under an inverted microscope (Leica).
The Ca content in the aortic tissue section and cells were measured using the o-cresolphthalein coplexone method, as previously described [22 (link)]. Quantification of Ca (μg/mg protein) was normalized to the protein concentration, which was determined using the Bicinchoninic Acid Protein Assay Kit (P0012S, Beyotime).