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Novel Red Fluorescent Calcium Probes for Functional Analysis of GPCRs and Calcium Channel Targets
by George Yi, Haitao Guo
The intercellular calcium flux assay is widely used for monitoring GPCRs and calcium channels. In our previous work, Cal-520 AM has been developed as a new green fluorescent dye with a significantly improved signal to noise ratio and better intracellular retention than Fluo-3 AM and Fluo-4 AM. In this study, two new red fluorescent calcium indicators, Cal-590 AM and Cal-630AM, have been developed for monitoring calcium ions in GFP cell lines or multiplexed with green-fluorescent dyes. Cal-590 AM and Cal-630AM are much more sensitive than rhodamine calcium dyes (such as Rhod-2, AM). Instead of located mostly in mitochondria as for Rhod-2, Cal-590 and Cal-630 are retained in cytoplasm. When stimulated with bioactive compounds, the red fluorescence of Cal-590 and Cal-630 are greatly enhanced when binding intracellular calcium with no overlap with green fluorescence.
Cal 520 AM, Cal-590AM and Cal-630 AM have been developed for evaluating GPCR and calcium channel targets, as well as for screening their agonists and antagonists. They have the following features:
Original created on February 25, 2015, last updated on November 7, 2022
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Introduction
[Ca2+] Increase via Gq or calcium channel is measured by calcium dyes.
Experiments
- CHO-K1 and CHO-GFP cells were seeded overnight in 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate at 37oC incubator.
- Take out growth medium. Add 100 µL of 5 µg/ml Cal-520 AM, Cal-590 AM, Cal630 AM, Rhod-2 AM, or Fluo-4 AM with different dose of probenecid (PBC) to cells. Incubate the cells at 37oC for 1 hour, then remove the dye loading buffer and replace with 200 uL HH, at room temperature for 15 min.
- Add ATP (50µL/well) with FlexStation (Molecular Devices) to achieve the final indicated concentrations. Run calcium efflux experiments on FlexStation or take images with fluorescence microscope (Olympus IX71).
Emission Spectra of Cal-520, Cal-590, Cal-630, and Rhod-2 (calcium bound).
Response of endogenous P2Y receptor to ATP in CHO-K cells. Images were recorded with a fluorescence microscope (Olympus IX71) before and after adding 10 µM ATP (final in the well) using FITC channel (Cal-520 AM), TRITC channel (Cal-590 AM) and Texas Red Channel (Cal-630 AM).
ATP-stimulated calcium response on CHO-GFP cells incubated with Cal-590 AM, Rhod-2 AM under the same conditions.10 µM ATP (final concentration in the well) was added by FlexStation (Molecular Devices).
ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with different Ca2+ indicators under the same conditions. ATP (50 µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations. (A: Cal-520 AM with 1.0 mM PBC and Fluo-4 AM with 2.5 mM PBC; B: Cal-520 AM and Fluo-4 AM without PBC; C: Cal-590 AM with 1.0 mM PBC and Rhod-2 AM with 2.5 mM PBC; D: Cal630 AM with 1.0 mM PBC).
Summary
Cal 520 AM, Cal-590AM and Cal-630 AM have been developed for evaluating GPCR and calcium channel targets, as well as for screening their agonists and antagonists. They have the following features:
- High S/N ratio: significantly higher S/N ratio than any other commercially available fluorescent Ca2+ indicators.
- Enable multicolor detection from green to red fluorescence.
- Improved intracellular retention: Minimal probenecid is required.
- Located in cytoplasm with minimal distribution in organelles.
Original created on February 25, 2015, last updated on November 7, 2022
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