InSituCellDeathDetectionKit,POD 

TUNEL法原位检测细胞凋亡试剂盒

Forlifescienceresearchonly.Notforuseindiagnosticprocedures.

本产品仅用于科研用途。

ProductNo.：11684817910

PackSize：1kitforupto50tests             

Overview

Sensitive:Themaximumintensityoflabeling(cellstaining)ofapoptoticcellsishigherthanthenicktranslationmethod

Fast:Theuseoffluorescein-dUTPallowsanalysisofthesamplesdirectlyaftertheTUNELreaction,butbeforetheadditionofthesecondarydetectionsystem

Convenient:Thedirectlabelingprocedureusingfluorescein-dUTPallowsverificationoftheefficiencyoftheTUNELreactionduringtheassayprocedure

Accurate:Identificationofapoptosisatamolecularlevel(DNA-strandbreaks)andidentificationofcellsattheveryearlystagesofapoptosis

Flexible:Nosubstrateincluded;providestheopportunitytoselectthestainingprocedureofchoice

Contents

EnzymeSolution(TdT)

LabelSolution(fluorescein-dUTP)

ConverterPOD(anti-fluoresceinantibody-POD),ready-to-use

Application

Qualitativedetectionofapoptosisatthesingle-celllevelbylightmicroscopy.

Details

WidelyusedmethodstodetermineapoptosisincludetheanalysisofthegenomicDNAbyagarose-gelelectrophoresisandDNAfragmentationassaysbasedon ³H-thymidineand,alternatively,5-Bromo-2-deoxy-uridine.Themethodsinvolvetheseparationoffragmented,lowmolecular-weightDNAfromunfragmented,highmolecular-weightDNAinagivencellpopulation.Thus,thesemethodsdonotprovideinformationaboutthefateofanindividualcellinagivencellpopulationor,particularly,intissuesections.Alternatively,individualapoptoticcellsmaybemicroscopicallyrecognizedbecauseofthecharacteristicappearanceofnuclearchromatincondensationandfragmentation,butthismethodissubjectiveandlimitedtoarelativelynarrowtimewindowwhenthemorphologicalchangesareatamaximum.
ThehallmarkofapoptosisisDNAdegradation,whichinearlystagesisselectivetotheinternucleosomalDNAlinkerregions.TheDNAcleavagemayyielddouble-strandedandsingle-strandedDNAbreaks(nicks).Bothtypesofbreakscanbedetectedbylabelingthefree3-OHterminiwithmodifiednucleotides(e.g.,biotin-dUTP,DIG-dUTP,fluorescein-dUTP)inanenzymaticreaction.Theenzymeterminaldeoxynucleotidyltransferase(TdT)catalyzesthetemplate-independentpolymerizationofdeoxyribonucleotidestothe3-endofsingle-anddouble-strandedDNA.ThismethodhasalsobeentermedTUNEL(TdT-mediateddUTP-X nick end labeling).Alternatively,free3-OHgroupsmaybelabeledusingDNApolymerasesbythetemplate-dependentmechanismcallednicktranslation.However,theTUNELmethodisconsideredtobemoresensitiveandfaster.

Samplematerial: Cytospinandcellsmearpreparations,adherentcellsgrownonslides,andfrozenandparaffin-embeddedtissuesections.
 

Principle

The InSitu CellDeathDetectionKit,POD isbasedonthedetectionofsingle-anddouble-strandedDNAbreaksthatoccurattheearlystagesofapoptosis.
Apoptoticcellsarefixedandpermeabilized.Subsequently,thecellsareincubatedwiththeTUNELreactionmixturethatcontainsTdTandfluorescein-dUTP.Duringthisincubationperiod,TdTcatalyzestheadditionoffluorescein-dUTPatfree3-OHgroupsinsingle-anddouble-strandedDNA.Afterwashing,thelabelincorporatedatthedamagedsitesoftheDNAismarkedbyananti-fluoresceinantibodyconjugatedwiththereporterenzymeperoxidase.Afterwashingtoremoveunboundenzymeconjugate,thePODretainedintheimmunecomplexisvisualizedbyasubstratereaction.