Calcium deposition quantification

The calcium deposition of VSMCs was measured, and the cells were rinsed multiple times with 1× PBS before being decalcified overnight with 0.6 N HCl. Colorimetrically, the calcium concentration in the supernatant was measured using the o-cresolphthaleincomplexone technique (Bioassay System, Cat. DICA500). The remaining cells were washed in 1× PBS and lysed in lysis buffer; total protein content was determined using the BCA technique (Pierce; Thermo Scientific, USA). Total calcium content was standardized by total protein level for each culture. In addition, a section of the aorta was weighed and decalcified with 0.6 N HCl for calcium determination in aortic tissues. Calcium content was measured using the technique described above, and the results were expressed as calcium per mg of dried aortic tissues.