Screen Quest™ Rhod-4 No Wash Calcium Assay Kit

Screen Quest™ Rhod-4 No Wash Calcium Assay Kit is a homogeneous fluorescence-based kit for monitoring intracellular calcium mobilization in cell-based microplate assays.

Calcium-responsive fluorescence: Rhod-4™ NW fluorescence increases upon calcium binding, providing a readout of intracellular calcium changes
Red fluorescence detection: Supports calcium response measurements with recommended fluorescence reader settings of Ex/Em = 540/590 nm
No-wash microplate format: Supports homogeneous assays in 96-well and 384-well plates and adaptation to automated screening
Research applications: Suitable for screening calcium-coupled GPCRs and evaluating calcium channel–associated responses

Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Rhod-4 NW Assay kit and Rhod-2 AM<strong>.</strong> HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well plate. 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 (25µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations. The EC<sub>50</sub> of Carbachol using Rhod-4 NW is about 0.6 µM.

Protocol

Catalog	Size	Price
36333	1 Plate	Price
36334	10 Plates	Price
36335	100 Plates	Price

Overview

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.

Components

Catalog 36333 (1 Plate)

Component A: Rhod-4™ NW	1 vial, lyophilized
Component B: 10X Pluronic® F127 Plus	1 bottle (1 mL)
Component C: HHBS (Hanks’ buffer with 20 mM Hepes)	1 bottle (9 mL)

Catalog 36334 (10 Plates)

Component A: Rhod-4™ NW	1 vial, lyophilized
Component B: 10X Pluronic® F127 Plus	10 bottles (1 mL/bottle)
Component C: HHBS (Hanks’ buffer with 20 mM Hepes)	1 bottle (100 mL)

Catalog 36335 (100 Plates)

Component A: Rhod-4™ NW	10 vials, lyophilized
Component B: 10X Pluronic® F127 Plus	10 bottles (10 mL/bottle)
Component C: HHBS (Hanks’ buffer with 20 mM Hepes)	Not included

References

View all 34 references: Citation Explorer

Fluorescence absorbance inner-filter decomposition: the role of emission shape on estimates of free Ca(2+) using Rhod-2

Authors:

Territo PR, Heil J, Bose S, Evans FJ, Balaban RS.

Journal:

Appl Spectrosc (2007): 138

Protein kinase C and myocardial calcium handling during ischemia and reperfusion: lessons learned using Rhod-2 spectrofluorometry

Authors:

Stamm C, del Nido PJ.

Journal:

Thorac Cardiovasc Surg (2004): 127

Novel fluo-4 analogs for fluorescent calcium measurements

Authors:

Martin VV, Beierlein M, Morgan JL, Rothe A, Gee KR.

Journal:

Cell Calcium (2004): 509

Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux

Authors:

Bednar B, Cunningham ME, Kiss L, Cheng G, McCauley JA, Liverton NJ, Koblan KS.

Journal:

J Neurosci Methods (2004): 247

Cytosolic calcium in the ischemic rabbit heart: assessment by pH- and temperature-adjusted rhod-2 spectrofluorometry

Authors:

Stamm C, Friehs I, Choi YH, Zurakowski D, McGowan FX, del Nido PJ.

Journal:

Cardiovasc Res (2003): 695

Spectral properties

Excitation (nm)	523
Emission (nm)	551
Quantum yield	0.1 ¹

Usage and storage

Intended use

Research Use Only (RUO)

Instrument settings

Fluorescence microplate reader
Excitation	540 nm
Emission	590 nm
Cutoff	570 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode/Programmable liquid handling

Other instruments	ArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLux

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Page updated on October 17, 2026