Effect of Ca × P ion products on serum depletion-induced calcification. Confluent cells were incubated in DMEM without serum at various levels of Ca × P ion products for 2 hrs. The culture dishes were washed twice with TBS and then suspended in 0.5 ml 0.1 N HCl. The extracts were centrifuged in a microfuge for 10 min at the top speed to collect the supernatants. Each set of three replicate experiments covered the indicated range of ion products. Calcification in cell culture was expressed as μmol of Ca and P deposited per ml of 0.1 N HCl extracts. Each data point for Ca and P represents Mean ± S.D. calculated from the average of duplicates in each experiment. The contributions of endogenous Ca and P in serum and the culture media to the ion products were all taken into considerations. (A) Ca concentration was kept at 1.80 mM whereas various concentrations of P were used. (B) P concentration was kept at 1.87 mM whereas various concentrations of Ca were used.