| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 492/514 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | ADMEandTox MDRResearch |
| Related | CellMetabolism CellFunctionalAnalysis BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/90µLfora96-wellplateor10,000to20,000cells/well/20µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinculturemediumat100,000-200,000cells/well/90µLfora96-wellpoly-Dlysineplateor25,000-50,000cells/well/20µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.Preparedye-loADIngsolution:
2.1 Thawallthekitcomponentsatroomtemperaturebeforeuse.
2.2 MakeMDRsensorstocksolution:Add20µL(Cat.#36340-1plate)or200µL(Cat.#36341-10plates)ofDMSO(ComponentB)intoMDRsensor(ComponentA),andmixthemwell.
Note:20µLofMDRsensorstocksolutionisenoughforoneplate.Un-usedMDRsensorstocksolutioncanbealiquotedandstoredat<-20oCforonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcyclesandmoisture.
2.3 MakeMDRdye-loadingsolutionforonecellplate:Add20µLofMDRsensorstocksolution(fromStep2.2)into10mLofAssayBuffer(ComponentC),andmixthemwell.TheMDRdye-loadingsolutionisstableforatleast2hoursatroomtemperature.
3.RunMDRassay:
3.1 Treatcellswithtestcompoundsbyadding10µLof10X(96-wellplate)or5µLof5X(384-wellplate)compoundsintocompoundbuffer(suchasPBSorHHBS).Forblankwells(mediumwithoutthecells),addthecorrespondingamountofcompoundbuffer.
Note:Itisnotnecessarytowashcellsbeforeaddingcompound.However,iftestedcompoundsareserumsensitive,growthmediumandserumfactorscanbeaspiratedawaybeforeaddingcompounds.AddthesamevolumeofHHBSintothewells(suchas90µLfora96-wellplateor20µLfora384-wellplate)afteraspiration.Alternatively,cellscanbegrowninserum-freemedia.
3.2 Incubatethecellplateatroomtemperatureorina37oC,5%CO2incubatorforatleast15minutesoradesiredperiodoftime.
3.3 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofMDRdye-loadingsolution.
3.4 Incubatethedye-loadingplateatroomtemperaturefor1hour,protectedfromlight.(Theincubationtimecouldbefrom15mintoovernight.Wegottheoptimalresultswiththeincubationtimelessthan4hours.)
Note1:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
Note2:DONOTwashthecellsafterloading.
Note3:Fornon-adherentcells,itisrecommendedtocentrifugethecellplateat800rpmfor2minuteswithbrakeoffafterincubation.
3.5 MonitorthefluorescenceintensityatEx/Em=490/525nmwithbottomreadmode.
| References&Citations |  PrinterFriendlyVersion |
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