Alizarin Red Staining: A Technique to Visualize Mineralization by Cultured Osteoblasts

Aspirate culture medium from primary osteoblasts, 14 days after the addition of osteogenic mineralization medium. Rinse twice with 0.5 milliliters of 1X PBS. Then, fix the cells by adding 0.5 milliliters of 10% buffered formalin solution, and incubating at room temperature for 10 minutes. After 10 minutes, aspirate the 10% buffered formalin with a single-use pipette, and wash twice with 0.5 milliliters of ultrapure water. Then, add 250 microliters of 40 millimolar Alizarin Red S staining solution to the fixed osteoblasts, and incubate the plate at room temperature for 10 minutes on a shaker at 100 shakes per minute.

Following the incubation with staining solution, aspirate the staining solution with a single-use pipette, and rinse with 1 milliliter of ultrapure water. Repeat this step 5 to 10 times until the rinsing solution comes off clear to remove non-specific staining. After aspirating the final wash, add 1 milliliter of cold PBS, and incubate the plate at room temperature for 10 minutes on a shaker as before. Next, transfer the stained beta-tricalcium phosphate disc to a new well, and scan the plates with a flatbed scanner to record mineralization.