Fluo 8 calcium assay kit

To characterize the effects of LPC and ryanodine on the calcium metabolism of paVICs, a Fluo-8 calcium assay kit (Abcam) was used to measure changes in cytoplasmic calcium in paVICs after introduction of those compounds to the medium. Cells were seeded onto a 96-well plate and allowed to attached overnight. The next day, the cells were loaded with the Fluo-8 dye for 30 minutes at 37°C then 30 min at room temperature, per the manufacturer’s directions. After loading, a Tecan 200 Infinite plate reader was used to pipette reagents to the final concentration indicated. Carbachol, which induces calcium flux in many cells types due to muscarinic agonism was used as a positive control. HEPES-buffered Hank’s balanced salt solution was used as a solvent for the reagents or as a negative control. After addition of the reagent, fluorescent intensity was recorded every second, and results were plotted as the maximum intensity after reagent addition minus the average intensity before addition of the reagent being tested. One biological replicate and 6–8 technical replicates were used.