Inducing and Modulating VIC Calcification

On day 0, paVICs were seeded into 48-well plates in basal media containing 1% BGS at 50,000 cells cm⁻², unless otherwise noted. On day 2, the experimental media conditions were added, and thereafter media was exchanged every 48 hours, keeping the experimental conditions, until the end of the experiment, at which point the cells were prepared for one of the assays described in the following sections. Four experimental media conditions were used: basal media, basal media supplemented with 10 μM LPC (Sigma Aldrich) to induce VIC calcification as previously described [20 (link)], basal media supplemented with ryanodine (Abcam) to modulate the activity of the RyR, and basal media supplemented with both ryanodine and LPC. LPC and ryanodine were first dissolved in absolute ethanol (Sigma Aldrich) before addition to the cell culture media, and an equivalent amount of ethanol was added to the basal media as a vehicle control. In some experiments, 10 nM ryanodine was used to increase the activity of the receptor, while in others 150 μM ryanodine was used to block the RyR activation [25 (link)].