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 |
1. Cases-GonzalezCE,FrancoS,MartinezMA,Menendez-AriasL.(2007)MutationalPatternsAssociatedwiththe69InsertionComplexinMulti-drug-resistantHIV-1ReverseTranscriptasethatConferIncreasedExcisionActivityandHigh-levelResistancetoZidovudine.JMolBiol,365,298.
2. O"ConnorR,O"LearyM,BallotJ,CollinsCD,KinsellaP,MagerDE,ArnoldRD,O"DriscollL,LarkinA,KennedyS,FennellyD,ClynesM,CrownJ.(2007)AphaseIclinicalandpharmacokineticstudyofthemulti-drugresistanceprotein-1(MRP-1)inhibitorsulindac,incombinationwithepirubicininpatientswithadvancedcancer.CancerChemotherPharmacol,59,79.
3. AdamsM,EfferthT,BauerR.(2006)Activity-guidedisolationofscopoletinandisoscopoletin,theinhibitoryactiveprinciplestowardsCCRF-CEMleukaemiacellsandmulti-drugresistantCEM/ADR5000cells,fromArtemisiaargyi.PlantaMed,72,862.
4. AnneseV,LatianoA,RossiL,BossaF,DamonteG,DallapiccolaB,SerafiniS,PierigeF,AndriulliA,MagnaniM.(2006)Thepolymorphismofmulti-drugresistance1gene(MDR1)doesnotinfluencethepharmacokineticsofdexamethasoneloadedintoautologouserythrocytesofpatientswithinflammatoryboweldisease.EurRevMedPharmacolSci,10,27.
5. BestB,HaubrichR.(2006)Tipranavir:aproteaseinhibitorformulti-drugresistantHIV-1.ExpertOpinInvestigDrugs,15,59.
6. BiancofioreG,TasciniC,BisaM,GemignaniG,BindiML,LeonildiA,GiannottiG,MenichettiF.(2006)Colistin,meropenemandrifampininacombinationtherapyformulti-drug-resistantAcinetobacterbaumanniimultifocalinfection.MinervaAnestesiol.
7. BrouwerCP,BogaardsSJ,WulferinkM,VeldersMP,WellingMM.(2006)Syntheticpeptidesderivedfromhumanantimicrobialpeptideubiquicidinaccumulateatsitesofinfectionsanderadicate(multi-drugresistant)Staphylococcusaureusinmice.Peptides,27,2585.
8. CartaA,LorigaM,PirasS,PagliettiG,LaCollaP,BusoneraB,ColluG,LoddoR.(2006)Synthesisofvariouslysubstituted3-phenoxymethylquinoxalin-2-onesandquinoxalinescapabletopotentiateinvitrotheantiproliferativeactivityofanticancerdrugsinmulti-drugresistantcelllines.MedChem,2,113.
9. ChenSH,YangCP,ChiuCH,ChiaJH,HuangIA,JaingTH.(2006)Fulminantsepticaemiacausedbymulti-drug-resistantStreptococcusmitisfollowingunrelatedcordbloodtransplantation.AnnTropPaediatr,26,247.
10. CobboldRN,RiceDH,DavisMA,BesserTE,HancockDD.(2006)Long-termpersistenceofmulti-drug-resistantSalmonellaentericaSEROvarNewportintwodairyherds.JAmVetMedAssoc,228,585.
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