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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/Cell Meter™ Apoptotic and Necrotic Multiplexing Detection Kit II *Triple Fluorescence Colors*/22843/100 T
产品编号:22843
市  场 价:¥85560.00
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:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Cell Meter™ Apoptotic and Necrotic Multiplexing Detection Kit II *Triple Fluorescence Colors*/22843/100 T
商品介绍
Overview
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Ex/Em(nm)None/None
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryCellAnalysis
CellCytotoxicity
RelatedApoptosisandCytotoxicity
CellApoptosis
BiochemicalAssays
OurCellMeter™assaykitsareasetoftoolsformonitoringcellviABIlity.Thereareavarietyofparametersthatcanbeused.Thisparticularkitisdesignedtosimultaneouslymonitorapoptotic,necroticandhealthycells.Apoptosisisdescribedasanactive,programmedprocessofautonomouscellulardismantlingthatavoidselicitinginflammation.Inapoptosis,phosphatidylserine(PS)istransferredtotheouterleafletoftheplasmamembrane.Asauniversalindicatoroftheinitial/intermediatestagesofcellapoptosis,theappearanceofphosphatidylserineonthecellsurfacecanbedetectedbeforemorphologicalchangesareobserved.ThePSsensorusedinthiskithasredfluorescence(Ex/Em=630/650nm)uponbindingtomembranePS.Necrosishasbeencharacterizedaspassive,accidentalcelldeathresultingfromenvironmentalperturbationswithuncontrolledreleaseofinflammatorycellularcontents.Lossofplasmamembraneintegrity,asdemonstratedbytheabilityofamembrane-impermeableDNANuclearGreen™DCS1(Ex/Em=490/525nm)tolabelthenucleus,representsastraightforwardapproachtodemonstratelatestageapoptosisandnecrosis.Inaddition,thiskitalsoprovidesalivecellcytoplasmlabelingdyeCytoCalcein™Violet450(Ex/Em=405/450nm)forlabelinglivingcellcytoplasm.Thiskitisoptimizedtosimultaneouslydetectcellapoptosis(Red),necrosis(greenand/orred)andhealthycells(blue)withaflowcytometerorfluorescencemicroscope.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.PrepareandincubatecellswithApopxin™DeepRed:

1.1   Treatcellswithtestcompoundsforadesiredperiodoftime(4-6hoursforJurkatcellstreatedwithstaurosporine)toinduceapoptosis.

1.2    Centrifugethecellstoget1-5×105cells/tube.

1.3    ResUSPendcellsin200μLofAssayBuffer(ComponentB).

1.4    Add2μLofApopxin™Red(ComponentA)intothecells.

Optional1:Add1µLof200XNuclearGreen™DCS1(ComponentC)intothecellsfornecrosiscells.

Optional2:Add100µLofDMSOintothevialofCytoCalcein™Violet450(ComponentD)tohave200XCytoCalcein™Violet450stocksolution,andthenadd1µLintothecellsforhealthycellsstaining.

1.5    Incubateatroomtemperaturefor30to60minutes(protectedfromlight).

1.6   Add300μLofAssayBuffer(ComponentB)toincreasevolumebeforeanalyzingthecellswithaflowcytometerorfluorescencemicroscope(seeStep1.7below).

1.7   MonitorthefluorescenceintensityatEx/Em=630/660nmforapoptosis,490/520nmfornecrosis,and405/450nmforhealthycellsusingaflowcytometerorafluorescencemicroscope(SeeStep2or3below).

2.Analyzecellsusingaflowcytometer:

QuantifyApopxin™RedbindingbyusingtheFL4channel(Ex/Em=630/660nm),andmeasurethecellviabilityusingtheFL1channel(Ex/Em=490/520nm)whenNuclearGreen™DCS1isadded,and/orusingEx/Em=405/450nmwhenCytoCalcein™Violet450isaddedintothecells.

Note:TheflowcytometricanalysisofApopxin™bindingtoadherentcellsisnotroutinelytestedsincespecificmembranedamagemayoccurduringcelldetachmentorharvesting.However,methodsforutilizingAnnexinVforflowcytometryonadherentcelltypeshavebeenpreviouslyreportedbyCasiola-Rosenetal.andvanEngelendetal(seeRefs1and2).

3.Analyzecellsusingafluorescencemicroscope:

3.1   PipettethecellsuspensionfromStep1.5,rinse1-2timeswithassaybuffer,andthenresuspendthecellswithassaybuffer.Addthecellsonaglassslidethatiscoveredwithaglasscoversliporablackwall/clearbottom96-wellmicroplate.

Note:Foradherentcells,itisrecommendedtogrowthecellsdirectlyonacoverslip(orablackwall/clearbottom96-wellmicroplate).AfterincubationwithApopxin™DeepRed(Step1.5),rinse1-2timeswithassaybuffer,andthenaddassaybufferbacktothecoverslip(orablackwall/clearbottom96-wellmicroplate).Invertcoversliponaglassslideandvisualizethecells.Thecellscanalsobefixedin2%formaldehydeaftertheincubationwithApopxin™DeepRedandvisualizedunderamicroscope.

3.2   AnalyzetheapoptoticcellswithApopxin™DeepRedunderafluorescencemicroscopeusingtheCy5channel.MeasurethecellviabilityusingtheFITCchannelwhenNuclearGreen™DCS1isadded,and/orVioletchannelwhenCytoCalcein™Violet450isaddedintothecells.TheredstainingontheplasmamembraneindicatestheApopxin™DeepRedbindingtoPSoncellsurface.

References&Citations
CitationExplorer

Anthocyanin-richblackcurrantextractinhibitsproliferationoftheMCF10AhealthyhumanbreastepithelialcelllinethroughinductionofG0/G1arrestandapoptosis
Authors:NaokiNanashima,KayoHorie,MitsuruChiba,ManabuNakano,HayatoMaeda,ToshiyaNakamura
Journal:MolecularMedicineReports(2017):6134--6141

ClusterinsignalsviaApoER2/VLDLRandinducesmeiosisofmalegermcells
Authors:MuhammadAssadRiaz,AngelikaStammler,MareikeBorgers,LutzKonrad
Journal:AmericanJournalofTranslationalResearch(2017):1266

DetectingApoptosis,Autophagy,andNecrosis
Authors:JackColeman,RuiLiu,KathyWang,ArunKumar
Journal:ApoptosisMethodsinToxicology(2016):77--92


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