| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 535/560 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | IonChannels MembranePotentials |
| Related | BiochemicalAssays |
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<-20oCformorethan6monthsifthetubesaresealedtightly,avoidinglightandrepeatedfreeze-thawcycles.
Note2: ComponentBandCcanbestoredat4oCforconvenience.
2.2 Make1Xassaybufferbymixing1mLofComponentB(10XAssaybuffer)with9mLofComponentC(HHBS,notincludedinthekit#36001),mixthemwell.
Note: 10ml1Xassaybufferisenoughfor1plate,aliquotandstoreun-used1Xassaybufferat<-20oC,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 |  PrinterFriendlyVersion |
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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.
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