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FLIPR® Calcium Assays
Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCRs). Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Fluo-8® AM or Rhod-4™ AM which can cross cell membrane. Screen Quest™ Calcium Assay Kits provide an optimized assay method for monitoring GPCRs and calcium channels. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation.
Fluo-8® AM is the brightest calcium indicator available for HTS screening. The characteristics of the convenient 488 nm excitation, high sensitivity, and 100-250 times fluorescence increases (when it forms complexes with calcium) make Fluo-8® AM an ideal indicator for the measurement of intracellular calcium.
Fig. 1
Carbachol dose responses were measured in HEK-293 cells with Screen Quest™ Fluo-8® No Wash Calcium Assay Kit (blue curve, Cat# 36315) and Fluo-4 No Wash Calcium Assay Kit (red curve). HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using Screen Quest™ Fluo-8® No Wash Calcium Assay Kit or Fluo-4 No Wash Kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
Carbachol dose responses were measured in HEK-293 cells with Screen Quest™ Fluo-8® No Wash Calcium Assay Kit (blue curve, Cat# 36315) and Fluo-4 No Wash Calcium Assay Kit (red curve). HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using Screen Quest™ Fluo-8® No Wash Calcium Assay Kit or Fluo-4 No Wash Kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
Rhod-4™ AM is the brightest red calcium indicator available for HTS screening. Once inside the cell, the lipophilic blocking groups of Rhod-4™ AM are cleaved by non-specific cell esterase, resulting in a negatively charged fluorescent dye that stays inside cells, and its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with screening compounds, the receptor signals the release of intracellular calcium, which greatly increases the fluorescence of Rhod-4™. The characteristics of its long wavelength, high sensitivity, and >250 times fluorescence increases (when it forms complexes with calcium) make Rhod-4™ AM an ideal indicator for the measurement of intracellular calcium.
Fig. 2
ATP dose responses were measured in CHO cells with Screen Quest™ Fura-2 No Wash Calcium Assay Kit (Cat# 36320). CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The cells were incubated with 100 µL of Screen Quest™ Fura-2 No Wash Calcium Assay Kit for 1 hour at room temperature. ATP (50 µL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.
ATP dose responses were measured in CHO cells with Screen Quest™ Fura-2 No Wash Calcium Assay Kit (Cat# 36320). CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The cells were incubated with 100 µL of Screen Quest™ Fura-2 No Wash Calcium Assay Kit for 1 hour at room temperature. ATP (50 µL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.
Screen Quest™ Fura-2 No Wash Calcium Assay Kit provides the only ratiometric FLIPR® calcium assay commercially available for screening GPCRs and calcium channel targets. The Fura-2 kit uses excitation ratio of 340/380 nm, monitoring emission at 510 nm.
Document: 03.0076.150501r1
Last updated Tue Sep 09 2025
FLIPR® Calcium Assays
Fig. 1
Carbachol dose responses were measured in HEK-293 cells with Screen Quest™ Fluo-8® No Wash Calcium Assay Kit (blue curve, Cat# 36315) and Fluo-4 No Wash Calcium Assay Kit (red curve). HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using Screen Quest™ Fluo-8® No Wash Calcium Assay Kit or Fluo-4 No Wash Kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
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Fig. 2
ATP dose responses were measured in CHO cells with Screen Quest™ Fura-2 No Wash Calcium Assay Kit (Cat# 36320). CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar 96-well black wall/clear bottom plate. The cells were incubated with 100 µL of Screen Quest™ Fura-2 No Wash Calcium Assay Kit for 1 hour at room temperature. ATP (50 µL/well) was added by FlexStation® (Molecular Devices) to achieve the final indicated concentrations.
View in articleFull size image