2.10. Alizarin Red S Mineralization Staining

As described above, paVICs were seeded into 48-well plates at a density of 50,000 cells cm⁻², and the experimental media conditions were added. Osteogenic media consisting of basal media with 10 mM β-glycerophosphate (Sigma Aldrich) and 10 nM dexamethasone (Sigma Aldrich), which was previously shown to promote mineralization of paVICs in vitro [30], was used as a positive control. On day 8, the cells were stained with Alizarin Red S (ARS) to highlight mineralized regions, as described previously [31]. First, the media was removed from the wells and the cells were fixed with 4% paraformaldehyde (Fisher Scientific), for 15 min at room temperature. The wells were then washed 3× with distilled, deionized water (ddH₂O). The cells were then incubated with a 40 mM ARS (Sigma Aldrich), adjusted to a pH of 4.1, for 30 minutes at room temperature, before being washed three more times with ddH₂O. The stained wells were then imaged with phase contrast microscopy with a Nikon Ti-E inverted microscope. Four representative images were taken from each well, and the number and area of the nodules in each image were quantified with a custom CellProfiler pipeline. The sum of the mineralized area in the four images was summed and then normalized to the total well area. Three biological replicates and 4–6 technical replicates were used.