In-Situ-Cell-Death-Detection-Kit--TMR-red

(programmedcelldeath)atsinglecelllevel,basedonlabelingofDNAstrandbreaks(TUNELtechnology):Analysisbyfluorescencemicroscopyorflowcytometry.Cat.No.121567929101Kit(50tests)Storeat15to25°CVersionApril200612156792910a.fmPage1Wednesday,April26,20061:12PM:12PM3(showlabelwherepossible).Collectthesupernatantsfromthelabelingreactionsinatightlyclosed,non-breakablecontainerandindicatecontents.Discardasregulatedfortoxicwaste.KitContentsPleaserefertothefollowingtableforthecontentsofthekit.Vial/CapLabelContents1blueEnzymeSolution�Terminaldeoxynucleotidyltransferasefromcalfthymus(EC2.7.7.31),recom-binantinE.coli,instoragebuffer�10×conc.�5×50l2redLabelSolution�Nucleotidemixtureinreactionbuffer�1×conc.�5×550l12156792910a.fmPage3Wednesday,April26,20061:12PM(section3.2)�Cellsuspension(section3.2.1)�Adherentcells,cellsmearsandcytospinpreparations(section3.2.2.)�Cryopreservedtissue(section3.2.3.2)�Shaker�V-bot-tomed96-wellmicro-plate�Washingbuffer:Phosphatebufferedsaline(PBS*)�Fixationsolution:4%ParaformaldehydeinPBS,pH7.4,freshlyprepared�Permeabilisationsolution:0.1%TritonX-100in0.1%sodiumcitrate,freshlypre-pared(6)Paraffin-embeddedtissue(section3.2.3.1)�Xyleneandethanol(absolute,95%,90%,80%,70%,dilutedindoubledistilledwater)�Washingbuffer:PBS*�ProteinaseK*,nucleasefree,workingsolution:[10–20g/mlin10mMTris/HCl,pH7.4–8]Alternativetreatments�Permeabilisationsolution:(0.1%Triton1)X–100,0.1%sodiumcitrate),freshlypre-pared�Pepsin*(0.25%–0.5%inHCl,pH2)ortrypsin*,0.01NHCl,nucleasefree�0.1MCitratebuffer,pH6formicrowaveirradiationLabelingprotocol(section3.3)Positivecontrol(section3.3.1)�Micrococcalnucleaseor�DNaseIrecombinant*�Cellsuspensions(section3.3.2)�Adherentcells(section3.3.3)�Parafilmorcoverslips�HumidifiedchamberWashingbuffer:PBS*Difficulttissue(section3.3.4)�Plasticjar�Microwave�Humidifiedchamber�Citratebuffer,0.1M,pH6.0.�Washingbuffer:PBS*�Tris-HCl,0.1MpH7.5,containing3%BSA*and20%normalbovineserum1.2KitContents,continued12156792910a.fmPage4Wednesday,April26,20061:12PM5(mono-andoligonu-cleosomes)aswellassinglestrandbreaks(“nicks“)inhighmolecularweightDNA.ThoseDNAstrandbreakscanbeidentifiedbylabelingfree3’-OHter-miniwithmodifiednucleotidesinanenzymaticreaction.Fig.1:DNAoffixedcellslabeledbytheadditionofTMRreddUTPatstrandbreaksbyterminaltransferase.ApplicationTheInSituCellDeathDetectionKitisdesignedasaprecise,fastandsimple,non-radioactivetechniquetodetectandquantifyapoptoticcelldeathatsinglecelllevelincellsandtissues.Thus,theInSituCellDeathDetectionKitcanbeusedinmanydifferentassaysystems.Examplesare:�Detectionofindividualapoptoticcellsinfrozenandformalinfixedtissuesectionsinbasicresearch.�Determinationofsensitivityofmalignantcellstodruginducedapop-tosisincancerresearch.�Typingofcellsundergoingcelldeathinheterogeneouspopulationsby