AAT Bioquest

Novel Red Fluorescent Calcium Probes for Functional Analysis of GPCRs and Calcium Channel Targets

by George Yi, Haitao Guo


[Ca2+] Increase via Gq or calcium channel is measured by calcium dyes.
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.


  • 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

Emission Spectra of Cal-520, Cal-590, Cal-630, and Rhod-2 (calcium bound).

Response of endogenous P2Y receptor to ATP

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

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

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).



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 February 25, 2015
Tagged under: Calcium GPCR Analysis, Calcium Imaging, Calcium Mobilization