Thiskitprovidestwoapoptosismarkers,ournovelNucView™488Caspase-3SubstrateandAnnexinVconjugatedwithadye,forprofilingapoptoticcellsandstudyingtherelationoftwoimportantapoptosisevents,caspase-3activationandphosphatidylserine(PS)translocation.
AnnexinVconjugationforthesecondmarkerisavailablewiththefollowingdyes:Sulforhodamine101(TexasRed®)AnnexinV,CF™594AnnexinV,andCF™640RAnnexinV.
Therateofapoptosisvariesfromcelltocellevenwithinthesamepopulation.Asaresult,variousapoptoticeventsormarkersaccompanyingtheapoptoticprocessalsooccurdifferentlyamongcells.Thus,itisimportanttobeabletodetecttheseapoptoticeventsonanindividualcellbasis.TrADItionally,caspaseactivityhasbeendetectedeitherusingamembrane-impermeablefluorogenicenzymesubstratesuchasDEVD-R110,orafluorescently-labeledinhibitorsuchasaFLICAreagent.Intheformercase,celllysisisrequired,thusprecludingthedetectionofcaspaseactivityinlivecells.Inaddition,suchcaspaseassaysmeasureonlytheaveragecaspaseactivityofahighlyheterogeneouscellpopulationatagiventime.Inthelattercase,althoughaFLICAreagentcanenterlivecellstodetectcaspaseactivity,onlytheinitialfluorescentsignalfollowingtheapplicationofthereagentcantrulyreflecttheenzymeactivityorthestateoftheapoptoticcellsbecauseanydetectedsignalaftertheinitial“snapshot”willneedtoconsiderthepotentialinterferenceoftheinhibitortotheenzymeandtheapoptoticcellitself.
Unlikeconventionalcaspaseassays,NucView™488Caspase-3substratedetectscaspase-3activitywithinindividualwholecellsinanon-interferingmanner.ThesubstrateconsistsofafluorogenicDNAdyeandaDEVDsubstratemoietyspecificforcaspase-3.Thesubstrate,whichisbothnon-fluorescentandnonfunctionalasaDNAdye,rapidlycrossescellmembranestoenterthecytoplasm,whereitiscleavedbycaspase-3toformahigh-affinityDNAdyethatstainsthenucleusbrightgreen.Thus,theNucView™488caspase-3substrateisbi-functional,allowingdetectionofcaspase-3activityandvisualizationofapoptoticnuclearmorphology.
Thekitcontainsreagentssufficientfor50flowcytometryassays(200uLassayvolume).Thenumberoffluorescencemicroscopyassaysthatcanbeperformedwiththekitmayvarybasedonthesizeofculturevesselandstainingvolumeused.
SeeourfullselectionofNucView™substratesandkits.
NucView™enzymesubstratetechnologyiscoveredbyU.S.patents.
TexasRed®isaregisteredtrademarkofMolecularprobes.
References
Cen,etal.DEVD-NucView488:anovelclassofenzymesubstratesforreal-timedetectionofcaspase-3activityinlivecells.TheFASEBJournalpublishedonlineFebruary8,2008.
DownloadalistofNucViewreferences:ValidatedcelllinesforNucView488withreferences
Downloadafulllistofreferences:ValidatedcelllinesforNucView488withreferences
1.PLoSGenet(2014),doi:10.1371/journal.pgen.1004594
2.CancerGeneTherapy(2016),doi:10.1038/cgt.2016.18
