Quantifying Osteogenic Differentiation via Alizarin Red

At 2, 4 and 6 weeks, CPC disks with cocultured cells were washed with PBS, fixed with 10% formaldehyde, and stained with Alizarin Red S (ARS, Millipore, Billerica, MA) for 20 min. ARS stains calcium-rich deposits synthesized by cells into a red color (Chen et al., 2012 (link), 2013 (link)). An osteogenesis assay (Millipore) was used to extract the stained minerals and measure the ARS concentration at OD₄₀₅, following the manufacturer’s instructions. The ARS standard curve was made with known concentration of the dye. CPC disks without cells (control) were also measured at the same time periods. The ARS concentration of control was subtracted from that of cell-seeded disks to yield the net mineral concentration synthesized by cells (Chen et al., 2012 (link), 2013 (link)).