Alizarin red s

Biomineralization was determined by an alizarin red S staining assay. Under the standard medium or OM for 14–21 days, cells were fixed with cold methanol for 30 min at 4°C and washed with distilled water. Subsequently, 1% alizarin red S (pH 4.2; Sigma) was added onto the samples and incubated for 10 min at room temperature, and the samples were rinsed twice with methanol to remove unbound alizarin red S. alizarin red S-stained mineralized matrices observed as bright red deposits were photographed under a Nikon digital camera.

Alizarin Red S (Sigma-Aldrich) was used in order to identify cultures’ mineralization 14 days after the induction of osteogenesis. At the end of the culturing time, cells were washed twice in PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min at RT, thus rinsed twice in ddH₂O and covered with a solution of Alizarin Red S 40 mM (pH 4.2) prepared in ddH₂O. Cultures were subsequently incubated for 40 min at 4 °C on an orbital shaker. After extensive washing with ddH₂O, specimen were air-dried before observation with an optical inverted microscope (Nikon).
After images acquisition Alizarin Red S concentration was measured by its solubilization. To this purpose, each well was treated with 400 μL of 10% acetic acid (Sigma-Aldrich) and incubated for 30 min at RT under shaking. Cells were then scraped from the plate, vortexed vigorously for 30 s, heated to 85 °C for 10 min, transferred on ice for 5 min and centrifuged at 20,000 rpm for 15 min at RT. After centrifugation, the supernatants were transferred into new eppendorf tubes, and the solution neutralized by adding 75 μL of 10% ammonium hydroxide. Sample absorbance was measured at 405 nm with a Multiskan^® FC microplate reader (Thermo Fisher Scientific, Carlsbad, CA, USA) and compared to a standard Alizarin Red S curve.