Screen Quest™ Membrane Potential Assay Kit Red Fluorescence

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Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

Screen Quest™ Membrane Potential Assay Kit *Orange Fluorescence*

Overview SDS Protocol

Membrane potential is the difference in voltage between the interior and exterior of a cell. The membrane potential allows a cell to function as a battery, providing power to operate a variety of "molecular devices" embedded in the membrane. In electrically excitable cells such as neurons, membrane potential is used for transmitting signals between different parts of a cell. Opening or closing of ion channels at one point in the membrane produces a local change in the membrane potential, which causes electric current to flow rapidly to other points in the membrane. Ion channels have been identified as important drug discovery targets. Our Screen Quest™ Membrane Potential Assay Kit is a homogeneous assay with fast read time. It uses our proprietary long wavelength membrane potential indicator to detect the membrane potential change that is caused by the opening and closing of the ion channels. The red fluorescence of the membrane potential indicator used in the kit has enhanced fluorescence upon entering cells and minimizes the interferences resulted from the screening compounds and/or cellular autofluorescence.

Platform

Fluorescence microplate reader

Excitation	620 nm
Emission	650 nm
Cutoff	630 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode/Programmable liquid handling

Other instruments

FDSS, FlexStation, NOVOStar

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells in growth medium or HHBS
Add MP dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
Incubate at room temperature or 37 ^oC for 1 hour
Monitor the fluorescence intensity at Ex/Em = 620/650 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

MP dye-loading solution:
Add 1 mL of 10X MP Sensor (Component A) into 9 mL of HHBS (Component B), and mix well. Note: The MP dye-loading solution is stable for at least 2 hours at room temperature. Note: 1 mL of 10X MP Sensor is enough for one plate. Unused 10X MP sensor (Component A) can be aliquoted and stored at < -20 ^oC for a few months, if stored properly. Avoid repeated freeze-thaw cycles. Note: HHBS (Component B) can be stored at 4 ^oC for convenience.

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

SAMPLE EXPERIMENTAL PROTOCOL

Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MP dye-loading solution into the cell plate. Note: If your screening compounds interfere with growth medium and serum factors, replace the growth medium with equal volume of HHBS buffer before adding the MP dye-loading solution. Alternatively, cells can be grown under serum-free conditions. Note: DO NOT wash the cells after dye loading.
Incubate the dye-loading plate in a 5% CO₂, 37 ^oC incubator for 30 to 60 minutes. Note: In some cases, 30 to 60 minutes room temperature incubation may work better.
Prepare the compound plates by using HHBS (Component B) or your desired buffer. Prepare the compound plates with HHBS or the desired buffer.
Monitor the fluorescence intensity at Ex/Em = 620/650 nm (bottom read). Note: It is important to run the signal test before the experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum instrument intensity counts.

Images

Figure 1. ATP dose response in HEK cells transiently transfected with P2X receptor. HEK cells transiently transfected with P2X receptor 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 the MP dye-loading solution in a 5% COâ‚‚, 37°C incubator for 60 minutes. ATP (50 µL/well) was added by FlexStation to achieve the final indicated concentrations. The fluorescence signal was measured with bottom read mode at Ex/Em = 620/650 nm (cutoff at 630 nm).

Citations

View all 1 citations: Citation Explorer

The short-chain fatty acid propionate increases glucagon and FABP4 production, impairing insulin action in mice and humans
Authors: Tirosh, Amir and Calay, Ediz S and Tuncman, Gurol and Claiborn, Kathryn C and Inouye, Karen E and Eguchi, Kosei and Alcala, Michael and Rathaus, Moran and Holl, undefined and er, Kenneth S and Ron, Idit and others, undefined
Journal: Science translational medicine (2019): eaav0120

References

View all 25 references: Citation Explorer

A novel high-throughput screening assay for HCN channel blocker using membrane potential-sensitive dye and FLIPR
Authors: Vasilyev DV, Shan QJ, Lee YT, Soloveva V, Nawoschik SP, Kaftan EJ, Dunlop J, Mayer SC, Bowlby MR.
Journal: J Biomol Screen (2009): 1119

A high-capacity membrane potential FRET-based assay for the sodium-coupled glucose co-transporter SGLT1
Authors: Weinglass AB, Swensen AM, Liu J, Schmalhofer W, Thomas A, Williams B, Ross L, Hashizume K, Kohler M, Kaczorowski GJ, Garcia ML.
Journal: Assay Drug Dev Technol (2008): 255

Miniaturization and HTS of a FRET-based membrane potential assay for K(ir) channel inhibitors
Authors: Solly K, Cassaday J, Felix JP, Garcia ML, Ferrer M, Strulovici B, Kiss L.
Journal: Assay Drug Dev Technol (2008): 225

A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry
Authors: Liu G, Amin S, Okuhama NN, Liao G, Mingle LA.
Journal: Histochem Cell Biol (2006): 283

Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators
Authors: Benjamin ER, Skelton J, Hanway D, Olanrewaju S, Pruthi F, Ilyin VI, Lavery D, Victory SF, Valenzano KJ.
Journal: J Biomol Screen (2005): 365

Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes
Authors: Felix JP, Williams BS, Priest BT, Brochu RM, Dick IE, Warren VA, Yan L, Slaughter RS, Kaczorowski GJ, Smith MM, Garcia ML.
Journal: Assay Drug Dev Technol (2004): 260

Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay
Authors: Jensen AA, Kristiansen U.
Journal: Biochem Pharmacol (2004): 1789

Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay
Authors: Jensen AA, Brauner-Osborne H.
Journal: Biochem Pharmacol (2004): 2115

Membrane potential fluorescence: a rapid and highly sensitive assay for nicotinic receptor channel function
Authors: Fitch RW, Xiao Y, Kellar KJ, Daly JW.
Journal: Proc Natl Acad Sci U S A (2003): 4909

A rapid assay for the brevetoxin group of sodium channel activators based on fluorescence monitoring of synaptoneurosomal membrane potential
Authors: David LS, Plakas SM, El Said KR, Jester EL, Dickey RW, Nicholson RA.
Journal: Toxicon (2003): 191