| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 635/660 |
| 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/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsinequalamountofHHBSandMPdye-loADIngsolution(seeStep2.2below)at125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.
2.PrepareMPdye-loadingsolution:
2.1 Thawonebottleof10XMPSensor(ComponentA),andonebottleofHHBS(ComponentB)atroomtemperaturebeforeuse.
Note1:1mLof10XMPSensor(ComponentA)isenoughforoneplate.Unused10XMPSensor(ComponentA)canbealiquotedandstoredat<-20oCforafewmonthsifthebottleissealedtightlyandkeptfromlight.Avoidrepeatedfreeze-thawcycles.
Note2:HHBS(ComponentB)canbestoredat4oCforconvenience.
2.2 MakeMPdye-loadingsolutionforonecellplatebyadding1mLof10XMPSensor(ComponentA)into9mLofHHBS(CompontB),andmixingthemwell.ThisMPdye-loadingsolutionisstableforatleast2hoursatroomtemperature.
3.RunMembranePotentialAssay:
3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofMPdye-loadingsolution(fromStep2.2)intothecellplate.
Note1:Ifyourscreeningcompoundsinterferewithgrowthmediumandserumfactors,replacethegrowthmediumwithequalvolumeofHHBSbufferbeforeaddingtheMPdye-loadingsolution.Alternatively,cellscanbegrownunderserum-freeconditions.
Note2:DoNOTwashthecellsafterdyeloading.
3.2 Incubatethedye-loadingplateina5%CO2,37oCincubatorfor30to60minutes.
Note:Insomecases,30to60minutesroomtemperatureincubationmayworkbetter.
3.3 PreparethecompoundplatesbyusingHHBS(ComponentB)oryourdesiredbuffer.
3.4 MonitorthefluorescenceintensityatEx/Em=620/650nm(bottomread).
Note:Itisimportanttorunthesignaltestbeforetheexperiment.Differentinstrumentshavetheirownintensityrange.Adjustthesignaltestintensitytothelevelof10%to15%ofthemaximuminstrumentintensitycounts.
| 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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