Cal-520™ provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilisation. Cal-520™ AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to
the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520™ AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal 520™ AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520™. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520™ AM an ideal indicator for the measurement of cellular calcium.The high S/N ratio and better intracellular retention make the Cal-520™ calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists. Compared to other Cal-520™ indicators, Cal-520FF™ has the lowest affinity to calcium with Kd ~ 10 uM.
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. x-Rhod-1 and x-Rhod-2 are commonly used as a red fluorescent calcium indicator.
However, x-Rhod-1 and x-Rhod-2 are only moderately fluorescent in live cells upon esterase hydrolysis, and has very small cellular calcium responses. Cal-630™ & Cal-590™ have been developed to improve x-Rhod-1 and x-Rhod-2 (respectively) cell loading and calcium response while maintaining the spectral wavelengths for x-Rhod-1 and x-Rhod-2. This makes Cal-630™ compatible with Texas Red® filter set. In CHO and HEK cells Cal-630™ AM has cellular calcium response that is much more sensitive than x-Rhod-1. In addition Cal-590™ is compatible with TRITC/Cy3® filter set. In CHO and HEK cells Cal-590™ AM has cellular calcium response that is much more sensitive than Rhod-2 AM.
The spectra of Cal-590™ & Cal-630™ are well separated from those of FITC, Alexa Fluor® 488 and GFP, making them ideal calcium probes for multiplexing intracellular assays with GFP cell lines or FITC/Alexa Fluor® 488 labelled antibodies.