详细信息:
Kitforthedetectionandquantificationofapoptosis(programmedcelldeath)atthesingle-celllevel,basedonlabelingofDNAstrandbreaks(TUNELtechnology):Analysisbyfluorescencemicroscopyorflowcytometry
Qualitativedetectionofapoptosisatthesingle-celllevelbyfluorescencemicroscopyandquantitativedetectionbyflowcytometry.
Samplematerial: CellsinsUSPension,cytospinandcellsmearpreparations,adherentcellsgrownonslides,andfrozenandparaffin-embeddedtissuesections.
WidelyusedmethodstodetermineapoptosisincludetheanalysisofthegenomicDNAbyagarose-gelelectrophoresisandDNAfragmentationassaysbasedon 3H-thymidineand,alternatively,5-Bromo-2-deoxy-uridine.Themethodsinvolvetheseparationoffragmented,lowmolecularweightDNAfromunfragmented,highmolecularweightDNAinagivencellpopulation.Thus,thesemethodsdonotprovideinformationaboutthefateofanindividualcellinagivencellpopulation,orparticularly,intissuesections.Alternatively,individualapoptoticcellsmaybemicroscopicallyrecognizedbecauseofthecharacteristicappearanceofnuclearchromatincondensationandfragmentation,butthismethodissubjectiveandlimitedtoarelativelynarrowtimewindowwhenthemorphologicalchangesareatamaximum.
ThehallmarkofapoptosisisDNAdegradation,whichinearlystages,isselectivetotheinternucleosomalDNAlinkerregions.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.
The InSitu CellDeathDetectionKitFluorescein isbasedonthedetectionofsingle-anddouble-strandedDNAbreaksthatoccurattheearlystagesofapoptosis.
ApoptoticcellsarefixedandpermeABIlized.Subsequently,thecellsareincubatedwiththeTUNELreactionmixturethatcontainsTdTandfluorescein-dUTP.Duringthisincubationperiod,TdTcatalyzestheadditionoffluorescein-dUTPatfree3-OHgroupsinsingle-anddouble-strandedDNA.Afterwashing,thelabelincorporatedatthedamagedsitesoftheDNAisvisualizedbyflowcytometryand/orfluorescencemicroscopy.
Sensitive: Thedirectlabelingprocedureusingfluorescein-dUTPreducesbackgroundlabeling
Fast: Theuseoffluorescein-dUTPallowsanalysisofthesamplesdirectlyaftertheTUNELreaction
Convenient: Nosecondarydetectionsystemrequired
Accurate: Identificationofapoptosisatamolecularlevel(DNA-strandbreaks)andidentificationofcellsattheveryearlystagesofapoptosis
EnzymeSolution(TdT),5vials
LabelSolution(fluorescein-dUTP),5vials
Roche
罗氏公司(Roche)致力于生命科学领域产品的开发,向用户提供优质的科研产品。在1998年收购德国宝灵曼(Boehringer Mannheim)公司后,产品涵盖了分子生物学,细胞学,免疫学,蛋白质组学,生物化学等多个研究领域,罗氏拥有PCR以及标记产品的全球专利,其DIG标记产品和细胞凋亡相关产品在业内享有极高的声誉。五十年来,罗氏应用科学部专注于生命科研领域产品的开发和推广,如今已经有超过2000种的科研产品在市场上销售,覆盖了生命科学研究领域的各个方面,为科研工作者提供完整的解决方案
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