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AAT Bioquest

Cal-630™ Calcium Indicators

emission spectra of Cal-520™ (Green), Cal-590™ (Orange) and Cal-630™ (Red)
Normalized emission spectra of Cal-520™ (Green), Cal-590™ (Orange) and Cal-630™ (Red).
X-Rhod-1 is commonly used as a red fluorescent calcium indicator. However, X-Rhod-1 is only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. In addition, X-Rhod-1 is mostly localized in mitochondria, thus giving low signal/background ratio. Cal-630™ has been developed to improve X-Rhod-1 cell loading and calcium response while maintaining the similar spectral wavelengths of X-Rhod-1, making it compatible with Texas Red® filter set. In CHO and HEK cells, the cellular calcium response of Cal-630™ is much more sensitive than that of X-Rhod-1. The maximum emission wavelength of Cal-630™ 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.



Fluorescence emission spectra of Cal-630™
ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with Cal-630™ AM

Left: Fluorescence emission spectra of Cal-630™ in solutions containing 0 to 39 µM free Ca2+. Right: ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with Cal-630™ AM (Cat# 20530). 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 10 µg/mL Cal-630™ AM with 2.0 mM probenecid was added into the cells, and the cells were incubated at 37 °C for 2 hours. ATP (50 µL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.


control
ATP

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 96-well black wall/clear bottom plate. 100 µL of 4 µM Cal-630™ AM (Cat# 20530) 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.

 

Table 1. Cal-630™ Calcium Detection Probes

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
20530Cal-630™ AM6086265x50 ug
20531Cal-630™ AM60862610x50 ug
20538Cal-630™, potassium salt6086265x50 ug
20535Cal-630™, sodium salt6086265x50 ug