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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™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit *Red Fluorescence*/20260/200 Tests
产品编号:20260
市  场 价:¥0.00
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:待定
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Cell Meter™ No-Wash Live Cell Caspase 3/7 Activity Assay Kit *Red Fluorescence*/20260/200 Tests
商品介绍
Overview
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Ex/Em(nm)405/600
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryCellAnalysis
CellApoptosis
RelatedApoptosisandCytotoxicity
Theactivationofcaspase3/7isimportantfortheinitiationofapoptosis.OurCellMeter™No-WashLiveCellCaspaseActivityAssayKitsarebasedonApoBrite™V600,ourrecentlydevelopedcell-permeablefluorogeniccaspasesubstrate,thefirstfluorogenicprobeforthedirectdetectionofcaspaseactivitiesinlivecells.ApoBriteV600consistsofthreemoietiesincludinga).maskedfluorophore,b).caspase-selectivepeptidefragment(DEVD),andc).cell-penetratingmoiety.Thecell-penetratingmoietycarriestheprobeintolivecells.Uponenteringlivecellsthecaspase-selectivepeptidefragmentiscleavedbyacaspasetoreleasethemaskedfluorophore.Theintensityofrecoveredfluorescenceisdirectlyrelatedtotheactivityofcaspasetobemeasured.Comparedtotheexistingcaspaseassaysinlivecells,ApoBrite™V600ismuchmorerobust,convenientandaccurate.ApoBrite™V600releasesafluorophorethathasalargeStokesshiftwithEx/Em~405/600,andcanbewellexcitedwithvioletlaserthatisinstalledmostofnewflowcytometers.ItdoesnotneedaDNAinteractiontobefluorescentasreportedforNucViewreagents.ItdoesnotinhibitcaspaseactivityasreportedfortheFMKpeptideprobes.AlthoughfluorescentFMKpeptideinhibitorsofcaspasesarewidelyusedfordetectingcaspaseactivitiesinlivecells,thistechnologyhasafewseverelimitations:a).FMKcaspaseinhibitorshavehighcytotoxicitysinceFMKpeptidesbindcovalentlytoactivecaspases;b).TheirreversIBLycovalentbindingofFMKpeptidestocaspasesinhibitscaspaseactivities,causingfalsepositiveapoptosis;c).FMKassayshaveextremelyhighbackground,andrequireintensivewashings,resultinginverylowthroughput;d).FMKpeptidesarenotstableinaqueoussolutions,andhavetobeusedimmediately.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.      Culturecellstoadensityoptimalforapoptosisinductionaccordingtoyourspecificinductionprotocol,butnottoexceed2x106cells/mLforsUSPensioncells.Foradherentcells,platecellsat30,000-80,000cells/well/96-wellplateovernight.

 

2.      Make1XApoBrite™V570Caspase3/7substrateworkingsolutionbyadding2µLof500XApoBrite™caspasesuV570Caspase3/7substrateDMSOstocksolution(ComponentA)into1mLHHBS(ComponentB).

Note:Theunused500XApoBrite™V570Caspase3/7substrateDMSOstocksolutionshouldbedividedassingleusealiquotandstoredat-20°C.

 

3.      Removegrowthmedium,andwashoncewithHHBS(ComponentB)orbufferofyourchoice.

 

4.      Add100µL/well/96wellplateof1XApoBrite™V570Caspase3/7substrateworkingsolution(fromStep2),andincubateat37ºC,5%CO2incubatorfor2hours.

 

5.      RemoveApoBrite™V570Caspase3/7substrateworkingsolutionandwashoncewithHHBS(ComponentB).

 

6.      AddgrowthmediumbacktotheApoBrite™V570caspase3/7loadedcells(fromStep5),andtreatthecellsasdesiredforapoptosis.Hereareafewexamplesfor inducingapoptosisincellculture:

 

1)TreatingJurkatcellswith2μg/mlcamptothecinfor3hours.

2)TreatingJurkatcellswith1μMstaurosporinefor3hours.Helacellsfor1hour.

3)TreatingHL-60cellswith4μg/mlcamptothecinfor4hours.

4)TreatingHL-60cellswith1μMstaurosporinefor4hours.

5)TreatingHelacellswith1μMstaurosporinefor1hour.

 

Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.

 

7.      Ifdesired,labelthecellswithaDNAstain(suchas7-AADfordeadcells)

 

8.      Monitorthefluorescenceintensitybyfluorescencemicroscopy,flowcytometer,orfluorescentmicroplatereaderatEx/Em=405/570nm(for7-AAD,Ex/Em=535/650nm)

8.1   Forflowcytometry(suspensioncells),monitorthefluorescenceintensityusingEx/Em=405/570nmfilters(FL3channelfor7-AADstaining).Gateonthecellsofinterest,excludingdebris.

 

8.2   Forfluorescencemicroscopy,observecellsunderafluorescencemicroscopeusingEx/Em=405/570nm orDAPIchannel(TRITCchannelfor7-AADstaining)

 

8.3   Forfluorescentmicroplatereader,monitorthefluorescenceintensityusingEx/Em=405/570nm(cutoffat540nm)bottomreadmode.

References&Citations
CitationExplorer

AngiopoietinsModulateSurvival,Migration,andtheComponentsoftheAng-Tie2PathwayofChronicLymphocyticLeukaemia(CLL)CellsInVitro
Authors:LuisMarioAguirrePalma,HannaFlamme,IrisGerke,Karl-AntonKreuzer
Journal:CancerMicroenvironment(2016):13--26

HypoxiaregulatesTRAILsensitivityofcolorectalcancercellsthroughmitochondrialautophagy.
Authors:GertrudKnoll,SebastianBittner,MariaKurz,JonathanJantsch,MartinEhrenschwender
Journal:Oncotarget(2016)

microRNA-186inhibitscellproliferationandinducesapoptosisinhumanesophagealsquamouscellcarcinomabytargetingSKP2
Authors:WeiHe,JianfangFeng,YanZhang,YuanyuanWang,WenqiaoZang,GuoqiangZhao
Journal:LaboratoryInvestigation(2016):317--324

Promotionofosteointegrationunderdiabeticconditionsbytantalumcoating-basedsurfacemodificationon3-dimensionalprintedporoustitaniumimplants
Authors:LinWang,XiaofanHu,XiangyuMa,ZhenshengMa,YangZhang,YizhaoLu,XiangLi,WeiLei,YafeiFeng
Journal:ColloidsandSurfacesB:Biointerfaces(2016):440--452

Roleofdelta-likeligand-4inchemoresistanceagainstdocetaxelinMCF-7cells
Authors:QWang,YShi,HJButler,JXue,GWang,PDuan,HZheng
Journal:Human&ExperimentalToxicology(2016):0960327116650006

Glucosepromotescellproliferation,glucoseuptakeandinvasioninendometrialcancercellsviaAMPK/mTOR/S6andMAPKsignaling
Authors:JianjunHan,LuZhang,HuiGuo,WeiyaZWysham,DarioRRoque,AdamKWillson,XiuguiSheng,ChunxiaoZhou,VictoriaLBae-Jump
Journal:Gynecologiconcology(2015):668--675

IL-7abrogatestheimmunosuppressivefunctionofhumandouble-negativeTcellsbyactivatingAkt/mTORsignaling
Authors:AndreaAllgäuer,ELISAbethSchreiner,FulviaFerrazzi,ArifBEkici,ArminGerbitz,AndreasMackensen,SimonVölkl
Journal:TheJournalofImmunology(2015):3139--3148

InsulinimprovesosteogenesisoftitaniumimplantsunderdiabeticconditionsbyinhibitingreactiveoxygenspeciesoverproductionviathePI3K-Aktpathway
Authors:LinWang,XiongZhao,Bo-yuanWei,YiLiu,Xiang-yuMa,JianWang,Peng-chongCao,YangZhang,Ya-boYan,WeiLei
Journal:Biochimie(2015):85--93

LGL1modulatesproliferation,apoptosis,andmigrationofhumanfetallungfibroblasts
Authors:HuiZhang,NeilBSweezey,FeigeKaplan
Journal:AmericanJournalofPhysiology-LungCellularandMolecularPhysiology(2015):L391--L402

t-BHQProvidesProtectionagainstLeadNeurotoxicityviaNrf2/HO-1Pathway
Authors:FangYe,XiaoyiLi,LiliLi,JingYuan,JunChen
Journal:Oxidativemedicineandcellularlongevity(2015)


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