Calcification Assay to Measure Calcium Concentration in Aortic Valve Interstitial Cells

Before beginning the assay, clean the biosafety hood with 70% ethanol, and warm the DMEM medium to 37 degrees Celsius. Next, seed 100,000 cells per condition into 6-well plates in complete DMEM, and culture at 37 degrees Celsius.

After 24 hours, replace the supernatant medium with the calcifying medium, and incubate the cells for 7 days at 37 degrees Celsius, replacing the medium on the third day.

After 7 days, measure the calcium concentration in a 96-well plate by using the Arsenazo III reagent, and recording the absorbance at 650 nanometers.