Alizarin red solution

At week three, cells were analyzed microscopically for extracellular matrix and bone nodule formation. At week four, the osteogenic cultures were stained for calcium deposition using 2% (w/v) Alizarin Red solution (Alfa Aesar). To do this, cultures were washed three times with PBS and fixed with 4% (w/v) PFA followed by the removal of PFA and washing with deionized water three times. The cultures were stained for 5 min using 200 μl Alizarin Red solution. The excess Alizarin Red solution was removed and the cultures were washed with deionized water. This was followed by incubation in deionized water for 2 h to remove the background staining. For calcium quantification, the stained cultures were washed three times with deionized water and Alizarin Red was extracted by the addition of 200 μl 10% (v/v) acetic acid (Sigma) for 30 min while shaking. The eluted stain was transferred to microfuge tubes and heated at 85°C for 10 min, cooled and neutralized with 10% (w/v) ammonium hydroxide (Sigma). The absorbance was read at 405nm on a plate reader (Infinite PRO, TECAN) (Gregory et al., 2004 (link)). Fold increase in molar concentration was compared to controls maintained in expansion media, basal media, OP or OI media (Gregory et al., 2004 (link)).