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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Screen Quest™ Fluorimetric MDR Assay Kit/36340/100 Tests
产品编号:36340
市  场 价:¥56560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$2828.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Screen Quest™ Fluorimetric MDR Assay Kit/36340/100 Tests
商品介绍
Overview
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Ex/Em(nm)492/514
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryADMEandTox
MDRResearch
RelatedCellMetabolism
CellFunctionalAnalysis
BiochemicalAssays
Tumorcellresistancetocytotoxicdrugsisconsideredoneofthemajorobstaclestosuccessfulchemotherapy.Sometumorsareinitiallyresistantandneverrespondtocytostaticdrugtreatment;othersinitiallyrespondwellbuteventuallyregrowandbecomeresistant.ThisphenomenonmayresultfromgeneticmutationsinducedbytheadmiNISTeredantitumoragent,ormayrepresenttheselectionofpreexistingresistantcellpopulationsinthemalignanttumor.Multi-drugresistance(MDR)isamajorfactorinthefailureofmanyformsofchemotherapy.InthepastfewyearsithasbecomewidelyacceptedthattheresistancetochemotherapycorrelateswiththeoverexpressionofatleasttwoATP-dependentdrug-effluxpumps.Thesecellmembraneproteins,calledP-glycoprotein(Pgp,MDR1),andmultidrug-resistance-associatedprotein(MRP1)aremembersoftheABCtransporterfamily.OurassaykitusesafluorescentMDRindicatorforassayingthesetwoMDRpumpactivities.Thishydrophobicfluorescentdyemoleculerapidlypenetratescellmembranesandbecomestrappedincells.Followingashortincubation,theintracellularfreedyeconcentrationcanincreasesignificantly.IntheMDR1and/orMRP1-expressingcellsthisdyeisextrudedbytheMDRtransporter,thusdecreasingthecellularfluorescenceintensity.However,whenitsextrusionisblockedbyanagentthatinterfereswiththeMDR1and/orMRP1pump-activity,itscellularfluorescenceintensityincreasessignificantly.OurMDRassaykitprovidesalltheessentialcomponentswithanoptimizedassaymethod.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomation.ThisassaykitisidealforhighthroughputscreeningofMDRpumpinhibitorsoridentifyingthecellsthathavehighlevelofMDRpumpactivities.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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
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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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