Cal-520™ for detection of intracellular calcium mobilization

Cal-520™ AM is our newest 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).
Cal-520™ provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization.

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 increases the fluorescence of Cal-520™.

The characteristics of its excellent cellular retention, high sensitivity and >100 times fluorescence enhancement make Cal-520™ AM an ideal indicator for the measurement of intracellular calcium. The high S/N ratio and better intracellular retention make the Cal-520™ calcium assay the most robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists. This new indicator has been successfully used for probenecid-sensitive cell lines and drug discovery targets.

Key Features of Cal-520™ Calcium Indicators (compared to Fluo-4)

• Excellent cellular retention, enabling Ca2+ assays of probenecid-sensitive GPCRs and Ca2+ channels.
• Robust, significantly higher S/N ratio than those of Fluo-4 AM and any other commercially available fluorescent Ca2+ assays.
• Convenient, essentially identical spectra to those of Fluo-4 and Fluo-8®.
• Versatile packing sizes to meet your special needs, 1 mg; 10×50 µg; HTS packages.

Figure 1. Responses of endogenous P2Y receptor to ATP in CHO-M1 cells without probenecid. CHO-M1 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 Fluo-4 AM (left), Cal 520™ AM (right) in HHBS was added into the wells, and the cells were incubated at 37 °C for 2 hours. The dye loading medium was replaced with 100 µL HHBS and 50 µL of 300 µM ATP were added. The cells were imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

Figure 2. ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without 2.5 mm probenecid) was added into the cells, and the cells were incubated at 37 °C for 2 hours. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.