A Functional Analysis of GPCR and Calcium Channel Targets using Cal 520 AM Ester

The Calcium influx assay is a preferred method for monitoring the activities of GPCR and calcium channels. Cal 520 AM is a new fluorescent calcium-sensitive dye developed for monitoring GPCR and calcium channel targets with a significantly improved signal to noise ratio and intracellular retention. Cal 520 AM ester is non-fluorescent. Once it enters the cells, the lipophilic AM blocking groups are cleaved by intracellular esterases, resulting in a negatively charged fluorescent dye that stays inside cells. When cells are stimulated with bioactive compounds, the receptor signals the release of intracellular calcium. As the dye binds to Ca2+ inside the cells, the fluorescence intensity of Cal 520 is greatly enhanced. In this study, the signal intensity and signal to background ratio of Cal 520 AM were evaluated with different receptor signaling pathways using several cell lines including HEK-293, CHO-M1 and Jurkat cells. Unlike the exisiting fluorescent calcium indicators (such as Fluo-3 AM and Fluo-4 AM) which are easily pumped out by organic-anion transporters, Cal 520 AM has much better cell retention ability in addition to its significantly higher signal to background ratio. It requires minimal amount of organic-anion transporter inhibitors (such as probenecid) present in the assay system. Organic-anion transporter inhibitors are often toxic to cells and also interfere with the activities of bioactive compounds to be screened. In conclusion, Cal 520 AM is an improved fluorescent indicator for the measurement of intracellular calcium. The high signal-to-noise ratio and good intracellular retention properties make the Cal 520 AM a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.