New Red Fluorescent Calcium Indicators for Functional Analysis of GPCRs and Ca2+ Channel Targets

The intercellular calcium flux assay is a widely used in monitoring signal transduction pathways and characterizing GPCRs agonists and antagonists. Most common calcium indicators are green fluorescent and difficult for using with GFP and YFP transfected cells or tissues, so red fluorescent calcium indicators are highly desirable. Although Rhod-2 is commonly used for multiplexing with GFP-transfected cells, the higher staining background and cellular localization (mostly in mitochondria) limits its applications and makes the rhodamine-based calcium dyes less sensitive. In this study, two new red fluorescent calcium indicators, Calbryte 590 and Calbryte 630, were developed for monitoring Ca2+ flux assays in living cells with Ex/Em= 580/592 nm and Ex/Em=608/624nm, which are red-shifted wavelength than Rhod-2 with no overlap with FITC filters. In this study, the performance of Calbryte 590 and Calbryte 630 was evaluated with different receptor signaling pathways using HEK-293, CHO-M1 and CHO-K1 cell lines with fluorescence microscope, microplate reader and flow cytometer. The fluorescence imaging data showed that Calbryte 590 and Calbryte 630 uniformly bind to cytosolic calcium ion. The microplate assay results showed that unlike Rhod-2, which requires the presence of organic anion transporter inhibitor (e.g. probenecid) to prevent the leakage of the indicators during dye loading, Calbryte 590 and Calbryte 630 requires minimal amount of probenecid and still has around 5 folds of response over background, which is more than twice higher compared to Rhod-2 assay (S/B ∼2 folds). Calbryte 630 flow cytometry assay results also showed ∼4 folds Ca2+ flux change with APC channel. In conclusion, Calbryte 590 and Calbryte 630 are robust red-shifted fluorescent indicators suitable for multiplexing Ca2+ flux assay in GFP- and YFP-transfected cells or with other green fluorescent dyes with improved cellular retention, brightness and S/B ratio.