MMB 282 Apoptosis Methods And Protocols

MMB 282 Apoptosis Methods And Protocols

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时间:2019-07-20

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1、METHODSINMOLECULARBIOLOGYMETHODSINMOLECULARBIOLOGYTMVolume282ApoptosisApoptosisMethodsMethodsandProtocolsandProtocolsEditedbyEditedbyHughJ.M.BradyHughJ.M.BradyMeasurementofApoptosisbyDNAFragmentation11MeasurementofApoptosisbyDNAFragmentationDemetriusMatassov,TerriKagan,JulieL

2、eblanc,MariannaSikorska,andZahraZakeriSummaryClassicalapoptoticcelldeathcanbedefinedbycertainmorphologicalandbio-chemicalcharacteristicsthatdistinguishitfromotherformsofcelldeath.Onesuchfeature,whichisahallmarkofapoptosis,isDNAfragmentation.Indyingcells,DNAiscleavedbyanendonu

3、cleasethatfragmentsthechromatinintonucleosomalunits,whicharemultiplesofabout180-bpoligomersandappearasaDNAladderwhenrunonanagarosegel.Here,wepresentcommonlyusedmethodssuchasconventionalagarosegelelectrophoresistoanalyzefragmentednucleiincells.Thevariousmethodsusedaredependent

4、ontheextentoffragmentationortheamountoffragmentednucleiinasample.DeterminingwhetheracellexhibitsDNAfrag-mentationcanprovideinformationaboutthetypeofcelldeathoccurringandthepathwaysactivatedinthedyingcell.KeyWords:Apoptosis;DNAfragmentation;electrophoresis;pulse-field;agaroseg

5、el.1.IntroductionThemethodsdescribedinthischapterdetectdamagedDNAincellsthathavefragmentedtheirgenomeduringprogrammedcelldeathorapoptosis.Apoptosisisanactiveregulatorycellularresponsetocertainstimuli,suchaslossoftrophicfactorsupport(serumdeprivation)orexposuretocytotoxicagent

6、s(chemotherapeuticFrom:MethodsinMolecularBiology,vol.282:ApoptosisMethodsandProtocolsEditedby:H.J.M.Brady©HumanaPressInc.,Totowa,NJ12Matassovetal.drugs),thatresultsinasomewhatstereotypedcelldeath(1–4),includingmembraneblebbing,nuclearcondensation,andthefor-mationofapoptoticbo

7、dies.Abiochemicalhallmarkofapoptosisisthecleavageofchromatinintosmallfragments,includingoligo-nucleosomes,thatwhenseeninelectrophoresedgelsaredescribedas“DNAladders”(5,6).DNAfragmentationwasfirstdocumentedbyWilliamsonin1970whenheobserveddiscreteoligomericfrag-mentsoccurringdu

8、ringcelldeathinprimaryneonatallivercultures(7).Afterthatdiscovery,Wi

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