Alizarin Red Staining and Calcium Quantification

Alizarin Red, or 1,2-dihydroxyanthraquinone was used to stain hydroxyapatite mineralized matrixes in cell monolayers producing a red-orange color. Alizarin Red powder (Sigma Aldrich) was dissolved in dH₂O to make a 40mM solution, and pH adjusted to 4.1–4.3 with 0.5% ammonium hydroxide. Cells were fixed with 10% (v/v) formaldehyde (Sigma Aldrich) at room temperature for 15 min. The monolayers were then washed twice with excess dH₂O. Alizarin Red solution was then added to each well and incubated at room temperature for 20 min. The unincorporated dye was then removed and the plates were washed 4 times with excess dH₂O. To extract and quantify the incorporated dye, 10% (v/v) acetic acid was added to each well. The cell layer mixture in acetic acid was then collected into eppendorfs, vortexed, and overlaid with mineral oil. The eppendorfs were heated to 85°C for 10 min and transferred to ice to cool. The samples were centrifuged at 20,000 × g for 15 min and the supernatants removed and neutralized with ammonium hydroxide (10% v/v). Colorimetric detection was then carried out at 405 nm and data expressed as absorbance.

Calcium content was measured using a calcium detection assay kit (Abcam, ab102505) according to manufacturer's instructions. Briefly, cells were decalcified overnight with 0.6 M hydrochloric acid (HCL). The calcium contents of the supernatants were then quantified using the 0-cresolphthalein method in which a chromogenic complex is formed between calcium ions and 0-cresolphthalein and then measured at 575 nm using a spectrophotometric plate reader (25). Calcium quantification was performed on days 0 and 21.