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Cal-590™ Calcium Indicators
In this issue
Fluorescent Calcium Indicators
Fluo-8® Calcium Indicators
Cal-520™ Calcium Indicators
Cal-590™ Calcium Indicators
Cal-630™ Calcium Indicators
Rhod-4™ Calcium Indicators
Ratiometric Calcium Indicators
FLIPR® Calcium Assays
AssayWise Letters 2015, Vol. 4(1)
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Rhod-2 AM is most commonly used among the red fluorescent calcium indicators. However, Rhod-2 AM is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses.
Cal-590™ has been developed to improve Rhod-2 AM cell loading and calcium response while maintaining the similar spectral wavelengths of Rhod-2 AM, making it compatible with TRITC/Cy3® filter set. In CHO and HEK cells, the cellular calcium response of Cal-590™ is much more sensitive than that of Rhod-2 AM. The spectra of Cal-590™ is well separated from those of FITC, Alexa Fluor® 488 and GFP, making it an ideal calcium probe for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies.
Left: The excitation and emission spectra of Cal-590™ in the presence of calcium chloride (5 mM). Right: Fluorescence emission spectra of Cal-590™ in solutions containing 0 to 39 µM free Ca2+.
Cal-590™ has been developed to improve Rhod-2 AM cell loading and calcium response while maintaining the similar spectral wavelengths of Rhod-2 AM, making it compatible with TRITC/Cy3® filter set. In CHO and HEK cells, the cellular calcium response of Cal-590™ is much more sensitive than that of Rhod-2 AM. The spectra of Cal-590™ is well separated from those of FITC, Alexa Fluor® 488 and GFP, making it an ideal calcium probe for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labeled antibodies.
ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-590™ AM (red curve, Cat# 20510) and Rhod-2, AM (blue curve) under the same conditions. CHO-K1 cells were seeded overnight at the cell density of 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom plate. 100 µL of 5 µg/mL Cal-590™ AM or Rhod-2 AM with 2.5 mM probenecid was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 µL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.
Responses of endogenous P2Y receptor to ATP in CHO-K1 cells. CHO-K1 cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. 100 µL of 4 µM Cal-590™ AM (Cat# 20510) in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 2 hours. The dye loading mediums were replaced with 100 µL HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using TRITC channel before and after adding 50 µL of 300 µM ATP.