In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction

Transfer the stained beta-tricalcium phosphate disks to a new well, and scan the plates with a flatbed scanner to record mineralization.

After image capture, add 250 microliters of 10% cetylpyridinium chloride solution and shake for 15 minutes to extract the Alizarin red S dye. Next, transfer the solution from the wells to individual 1.5-milliliter tubes, and centrifuge at 17,000 times g for 5 minutes at room temperature.

Dilute the extract with 10% cetylpyridinium chloride solution at a dilution ratio of 1:10 to 1:20 in a 1.5-milliliter tube. Then, transfer 300 microliters of each diluted sample into the wells of the 96-well plate. Include two blank wells containing only 10% cetylpyridinium chloride.

Next, prepare seven Alizarin red S reference standards ranging from 4 to 400 micromolar by diluting 40 millimolar Alizarin red S staining solution with 10% cetylpyridinium chloride solution to generate a standard curve.

Load 300 microliters of each standard into the plate. Lastly, read the absorbance of the samples, blanks, and reference standards at 520 nanometers.