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Screen Quest™ Rhod-4 No Wash Calcium Assay Kit
Medium Removal
Calcium flux assays are preferred methods in drug discovery for screening G protein coupled receptors (GPCR). Screen Quest™ Rhod-4 NW Calcium Assay Kit provides a homogeneous fluorescence-based assay for detecting the intracellular calcium mobilization. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with our proprietary Rhod-4 NW which can cross cell membrane. Rhod-4 is the brightest red calcium indicator available for HTS screening. Once inside the cell, the lipophilic blocking groups of Rhod-4™ 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 release of intracellular calcium, which greatly increase 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™ an ideal indicator for measurement of cellular calcium. This Screen Quest Rhod-4 NW Calcium Assay Kit provides an optimized assay method for monitoring G-protein-coupled receptors (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.
Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Rhod-4 NW Assay kit and Rhod-2 AM. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well plate. The growth medium was removed, and the cells were incubated with 100 µL of dye loading solution using the Screen Quest™ Rhod-4 NW calcium assay kit, or 100 µL of Rhod-2 AM solution (5 µM) for 1 hour at room temperature. Carbachol (25 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Cabachol by using Rhod-4 NW is about 0.8 µM.
Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Rhod-4 NW Assay kit and Rhod-2 AM. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well plate. The growth medium was removed, and the cells were incubated with 100 µL of dye loading solution using the Screen Quest™ Rhod-4 NW calcium assay kit, or 100 µL of Rhod-2 AM solution (5 µM) for 1 hour at room temperature. Carbachol (25 µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC50 of Cabachol by using Rhod-4 NW is about 0.8 µM.
protocol
Protocol
COO
COA
CatalogSize
Price
Quantity
363301 Plate
Price
3633110 Plates
Price
36332100 Plates
Price
 
Spectral properties
Excitation (nm)523
Emission (nm)551
Quantum yield
0.1
1
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
Instrument settings
Fluorescence microplate reader
Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling
Other instrumentsArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLux
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Page updated on September 25, 2025