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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/Annexin V-Cy5 conjugate/20066/100 Tests
产品编号:20066
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AAT Bioquest/Annexin V-Cy5 conjugate/20066/100 Tests
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Ex/Em(nm)651/660
MWN/A
CAS#N/A
SolventWater
StorageF/D/L
CategoryCellAnalysis
CellApoptosis
RelatedApoptosisandCytotoxicity
Annexinsareafamilyofproteinsthatbindtophospholipidmembranesinthepresenceofcalcium.AnnexinVisavaluabletoolforstudyingcellapoptosis.Itisusedasaprobetodetectcellswhichhaveexpressedphosphatidylserineonthecellsurface,afeaturefoundinapoptosisaswellasotherformsofcelldeath.Inapoptosis,PSistransferredtotheouterleafletoftheplasmamembrane.Theappearanceofphosphatidylserineonthecellsurfaceisauniversalindicatoroftheinitial/intermediatestagesofcellapoptosisandcanbedetectedbeforemorphologicalchangescanbeobserved.ThereareavarietyofparametersthatcanbeusedformonitoringcellviABIlity.AnnexinV-dyeconjugatesarewidelyusedtomonitorcellapoptosisthroughmeasuringthetranslocationofphosphatidylserine(PS).Cy5-AnnexinVprobeisoneofthemostpopularfluorescentdye-AnnexinVconjugatesusedformonitoringcellapoptosis.
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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareandincubatecellswithAnnexinVconjugates:

1.1   PrepareAnnexinV-bindingassaybuffer:10mMHEPES,140mMNaCl,and2.5mMCaCl2,pH7.4.

1.2   Treatcellswithtestcompoundsforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.

1.3   Centrifugethecellstoget1-5×105cells/tube.

1.4   ResUSPendcellsin200μLofAnnexinV-bindingassaybuffer(fromStep1.1).

1.5   Add2μLofAnnexinVconjugateintothecells.

Optional:AddadeadcellstainsuchasPropidiumIodideintothecellsfornecrosiscells.

1.6   Incubateatroomtemperaturefor30to60minutes,protectedfromlight.

1.7   Add300μLofAnnexinV-bindingassaybuffer(fromStep1.1)toincreasevolumebeforeanalyzingthecellswithaflowcytometerorfluorescencemicroscope(seeStep1.8below).

1.8   Monitorthefluorescenceintensitybyusingaflowcytometerorafluorescencemicroscope(SeeStep2or3below).

2.Analyzebyusingaflowcytometer:

QuantifyAnnexinVconjugatesbindingbyusingaflowcytometerwithappropriatedfilters.

Note:AnnexinVbindingflowcytometricanalysisonadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).

 

3.Analyzebyusingafluorescencemicroscope:

3.1   PipettethecellsuspensionfromStep1.6,rinse1-2timeswithAnnexinV-bindingassaybuffer(fromStep1.1),andthenresuspendthecellswiththeAnnexinV-bindingassaybuffer(fromStep1.1).Addthecellsonaglassslidethatiscoveredwithaglasscoverslip.

Note:Foradherentcells,itisrecommendedtogrowthecellsdirectlyonacoverslip.AfterincubationwithAnnexinVconjugate(Step1.6),rinse1-2timeswithAnnexinV-bindingassaybuffer(fromStep1.1),andaddAnnexinV-bindingassaybuffer(fromStep1.1)backtothecoverslip.Invertcoversliponaglassslideandvisualizethecells.Thecellscanalsobefixedin2%formaldehydeaftertheincubationwithAnnexinVconjugateandvisualizedunderamicroscope.

3.2   AnalyzetheapoptoticcellswithAnnexinVconjugateunderafluorescencemicroscopewithappropriatedfilters.

References&Citations
CitationExplorer

Resources-ApplicationNotesPhenotypicandEpigeneticMechanismofActionDeterminationsofHistoneMethylaseandDemethylaseInhibitorsusingDigitalWidefieldMicroscopy
Authors:BradLarson,PeterBanks
Journal:ExperimentalBIOLOGy(2017)

TargetedMagneticIntra-LysosomalHyperthermiaproduceslysosomalreactiveoxygenspeciesandcausesCaspase-1dependentcelldeath
Authors:PascalClerc,PaulineJeanjean,NicolasHalalli,MichelGougeon,BernardPipy,JulianCarrey,DanielFourmy,VéroniqueGigoux
Journal:JournalofControlledRelease(2017)


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