Overview

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Ex/Em(nm)	535/560
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	IonChannels MembranePotentials
Related	BiochemicalAssays

Membranepotentialisthedifferenceinvoltagebetweentheinteriorandexteriorofacell.Themembranepotentialallowsacelltofunctionasabattery,providingpowertooperateavarietyof"moleculardevices"embeddedinthemembrane.Inelectricallyexcitablecellssuchasneurons,membranepotentialisusedfortransmittingsignalsbetweendifferentpartsofacell.Openingorclosingofionchannelsatonepointinthemembraneproducesalocalchangeinthemembranepotential,whichcauseselectriccurrenttoflowrapidlytootherpointsinthemembrane.Ionchannelshavebeenidentifiedasimportantdrugdiscoverytargets.OurScreenQuest™MembranePotentialAssayKitisahomogeneousassaywithfastreadtime.Itusesourproprietarylongwavelengthmembranepotentialindicatortodetectthemembranepotentialchangethatiscausedbytheopeningandclosingoftheionchannels.Theredfluorescenceofthemembranepotentialindicatorusedinthekithasenhancedfluorescenceuponenteringcellsandminimizestheinterferencesresultedfromthescreeningcompoundsand/orcellularautofluorescence.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareCells

 

1.1   Foradherentcells,platecellsovernightingrowthmediumat40,000to80,000cells/well/100µlfor96-wellor10,000to20,000cells/well/25µlfor384-wellplates.

1.2   Fornon-adherentcells,centrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinequalamountofHHBSandMPdye-loADIngsolution(seeSteps2.3below)at125,000to250,000cells/well/100µlfor96-wellor30,000to60,000cells/well/25µlfor384-wellpoly-Dlysineplates. Centrifugetheplatesat800rpmfor2minuteswithbreakoffpriortotheexperiments

Note: Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforthemembranepotentialchange.

 

2.PrepareMPsensordye-loadingsolution(for1plate)

 

2.1   Thaw1vialofComponentA(MPsensor),ComponentB(10XAssaybuffer)andComponentC(HHBS)atroomtemperaturebeforeuse.

Note1: 15µlofcomponentA(MPsensor)isenoughfor1plate,un-usedComponentAcanbealiquotedandstoredat<-20^oCformorethan6monthsifthetubesaresealedtightly,avoidinglightandrepeatedfreeze-thawcycles.

Note2: ComponentBandCcanbestoredat4^oCforconvenience.

2.2   Make1Xassaybufferbymixing1mLofComponentB(10XAssaybuffer)with9mLofComponentC(HHBS,notincludedinthekit#36001),mixthemwell.

Note: 10ml1Xassaybufferisenoughfor1plate,aliquotandstoreun-used1Xassaybufferat<-20^oC,avoidlightandrepeatedfreeze-thawcycles.

 

2.3   MakeMPdye-loadingsolutionforonecellplatebyadding15µlofComponentA(MPsensor)into10mlof1Xassaybuffer(fromStep2.2),mixingthemwell. Thisworkingsolutionisstableforatleast2hoursatroomtemperature.

 

 

3. RunMembranePotentialAssay

 

3.1    Add100µL/well(96-wellplate)or25µL/well(384-wellplate)MPdye-loadingsolutionintothecellplate.

Note1: Ifyouscreencompoundsinterferewithgrowthmediumandserumfactors,thenreplacethegrowthmediumwithequalvolumeofHHBSbufferbeforeaddingtheMPdye-loadingbuffer.Alternatively,cellscanbegrowninserum-freeconditions.

Note2: DoNOTwashthecellsafterdyeloading.

3.2    Incubatethedye-loadingplateatcellincubatorfor30minutes.

Note:Insomecases,incubationatroomtemperaturefor30to60minmayworkbetter.

3.3   PreparethecompoundplatesbyusingHHBSoryourdesiredbuffer.

3.4   RunthemembranepotentialassaybymonitoringthefluorescenceatEx/Em=530/570nm.

Note:Itisimportanttorunthesignaltestbeforeyourexperiment. Differentinstrumentshavetheirownintensityrange.Adjustthesignaltestintensitytothelevelof10%to15%ofthemaximuminstrumentintensitycounts. Forexample,themaximumfluorescenceintensitycountforFLIPR-384is65,000,sotheinstrumentsettingsshouldbeadjustedtohaveitssignaltestintensityaround7,000to10,000.

References&Citations

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1.   VasilyevDV,ShanQJ,LeeYT,SolovevaV,NawoschikSP,KaftanEJ,DunlopJ,MayerSC,BowlbyMR.(2009)Anovelhigh-throughputscreeningassayforHCNchannelblockerusingmembranepotential-sensitivedyeandFLIPR.JBiomolScreen,14,1119.

2.   SollyK,CassadayJ,FelixJP,GarciaML,FerrerM,StruloviciB,KissL.(2008)MiniaturizationandHTSofaFRET-basedmembranepotentialassayforK(ir)channelinhibitors.AssayDrugDevTechnol,6,225.

3.   WeinglassAB,SwensenAM,LiuJ,SchmalhoferW,ThomasA,WilliamsB,RossL,HashizumeK,KohlerM,KaczorowskiGJ,GarciaML.(2008)Ahigh-capacitymembranepotentialFRET-basedassayforthesodium-coupledglucoseco-transporterSGLT1.AssayDrugDevTechnol,6,255.

4.   LiuCJ,PriestBT,BugianesiRM,DulskiPM,FelixJP,DickIE,BrochuRM,KnausHG,MiddletonRE,KaczorowskiGJ,SlaughterRS,GarciaML,KohlerMG.(2006)Ahigh-capacitymembranepotentialFRET-basedassayforNaV1.8channels.AssayDrugDevTechnol,4,37.

5.   BenjaminER,SkeltonJ,HanwayD,OlanrewajuS,PruthiF,IlyinVI,LaveryD,VictorySF,ValenzanoKJ.(2005)Validationofafluorescentimagingplatereadermembranepotentialassayforhigh-throughputscreeningofglycinetransportermodulators.JBiomolScreen,10,365.

6.   FelixJP,WilliamsBS,PriestBT,BrochuRM,DickIE,WarrenVA,YanL,SlaughterRS,KaczorowskiGJ,SmithMM,GarciaML.(2004)Functionalassayofvoltage-gatedsodiumchannelsusingmembranepotential-sensitivedyes.AssayDrugDevTechnol,2,260.

7.   JensenAA,Brauner-OsborneH.(2004)PharmacologicalcharacterizationofhumanexcitatoryaminoacidtransportersEAAT1,EAAT2andEAAT3inafluorescence-basedmembranepotentialassay.BiochemPharmacol,67,2115.

8.   JensenAA,KristiansenU.(2004)Functionalcharacterisationofthehumanalpha1glycinereceptorinafluorescence-basedmembranepotentialassay.BiochemPharmacol,67,1789.

9.   DavidLS,PlakasSM,ElSaidKR,JesterEL,DickeyRW,NicholsonRA.(2003)Arapidassayforthebrevetoxingroupofsodiumchannelactivatorsbasedonfluorescencemonitoringofsynaptoneurosomalmembranepotential.Toxicon,42,191.

10.   FitchRW,XiaoY,KellarKJ,DalyJW.(2003)Membranepotentialfluorescence:arapidandhighlysensitiveassayfornicotinicreceptorchannelfunction.ProcNatlAcadSciUSA,100,4909.

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