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Screen Quest™ Rhod-4 No Wash Calcium Assay Kit

Carbachol Dose Response was measured in HEK-293 cells with Screen Quest&trade; Rhod-4 NW Assay kit and Rhod-2 AM<strong>.</strong> HEK-293 cells were seeded overnight at 40,000 cells/100 &micro;L/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 &micro;L of dye-loading solution using the Screen Quest&trade; Rhod-4 NW calcium assay kit, or 100 &micro;L of Rhod-2 AM solution (5 &micro;M) for 1 hour at room temperature. Carbachol (25&micro;L/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 &micro;M.
Carbachol Dose Response was measured in HEK-293 cells with Screen Quest&trade; Rhod-4 NW Assay kit and Rhod-2 AM<strong>.</strong> HEK-293 cells were seeded overnight at 40,000 cells/100 &micro;L/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 &micro;L of dye-loading solution using the Screen Quest&trade; Rhod-4 NW calcium assay kit, or 100 &micro;L of Rhod-2 AM solution (5 &micro;M) for 1 hour at room temperature. Carbachol (25&micro;L/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 &micro;M.
Carbachol Dose Response was measured in HEK-293 cells with Screen Quest&trade; Rhod-4 NW Assay kit and Rhod-2 AM<strong>.</strong> HEK-293 cells were seeded overnight at 40,000 cells/100 &micro;L/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 &micro;L of dye-loading solution using the Screen Quest&trade; Rhod-4 NW calcium assay kit, or 100 &micro;L of Rhod-2 AM solution (5 &micro;M) for 1 hour at room temperature. Carbachol (25&micro;L/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 &micro;M.
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Spectral properties
Excitation (nm)523
Emission (nm)551
Quantum yield0.11
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Excitation (nm)
523
Emission (nm)
551
Quantum yield
0.11
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.

Platform


Fluorescence microplate reader

Excitation540 nm
Emission590 nm
Cutoff570 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Other instruments

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

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells in growth medium with 1-5% FBS
  2. Add Rhod-4 NW dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  3. Incubate at room temperature for 1 hour
  4. Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment. Do not add additional probenecid.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Rhod-4 NW stock solution:
Add 200 µL of DMSO into the vial of Rhod-4 NW (Component A) and mix well. Protect from light. Note: 20 µL of Rhod-4 NW stock solution is enough for one plate.  Note: Unused Rhod-4 NW stock solution can be aliquoted and stored at < -20 oC for more than one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles.

2. Assay Buffer (1X):
a) For Cat. # 36333 (1 plate kit) and # 36334 (10 plates kit) , make 1X assay buffer by adding 9 mL of HHBS (Component C) into 10X Pluronic® F127 Plus (1 mL, Component B), and mix them well.
b) For Cat. # 36335 (100 plates kit), make 1X assay buffer by adding 90 mL of HHBS (Not included) into 10X Pluronic® F127 Plus (10 mL, Component B), and mix them well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 1X assay buffer at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles.

PREPARATION OF WORKING SOLUTION

Rhod-4 NW dye-loading solution:
Add 20 µL of Rhod-4 NW stock solution into 10 mL of 1X assay buffer, and mix them well. Note: This working solution is stable for at least 2 hours at room temperature.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Rhod-4 NW dye-loading solution into the cell plate.

  2. Incubate the dye-loading plate in a cell incubator for 30 minutes, and then incubate the plate at room temperature for another 30 minutes. Note: If the assay requires 37 oC, perform the experiment immediately without further room temperature incubation. Note: If the cells can function well at room temperature for longer time, incubate the cell plate at room temperature for 1-2 hours.
     
  3. Prepare and add the compound plate with HHBS or your desired buffer.

  4. Run the calcium flux assay by monitoring the fluorescence intensity at Ex/Em = 540/590 nm.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)523
Emission (nm)551
Quantum yield0.11

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Screen Quest™ Fluo-4 No Wash Calcium Assay Kit495528820000.161

Images


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
Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions
Authors: Lannergren J, Westerblad H, Bruton JD.
Journal: J Muscle Res Cell Motil (2001): 265
Rhod-2 based measurements of intracellular calcium in the perfused mouse heart: cellular and subcellular localization and response to positive inotropy
Authors: MacGowan GA, Du C, Glonty V, Suhan JP, Koretsky AP, Farkas DL.
Journal: J Biomed Opt (2001): 23
Calibration of the calcium dissociation constant of Rhod(2)in the perfused mouse heart using manganese quenching
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Cell Calcium (2001): 217
Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory
Authors: Du C, MacGowan GA, Farkas DL, Koretsky AP.
Journal: Biophys J (2001): 549
Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death
Authors: Muriel MP, Lambeng N, Darios F, Michel PP, Hirsch EC, Agid Y, Ruberg M.
Journal: J Comp Neurol (2000): 297