1.Introduction

Nuclearmorphologychangescharacteristicofapoptosisappearwithinthecelltogetherwithadistinctivebiochemicalevent:theendonuclease-mediatedcleavageofnuclearDNA.Infact,formationofDNAfragmentsofoligonucleosomalsize(180-200bp)isanhallmarkofapoptosisinmanycelltypes.

ThepresentprotocolprovidesamethodforDNAseparationoffragmentedandintactDNAfractionsandfortheiranalysisbyagarosegelelectrophoresis.InapoptoticcellsspecificDNAcleavagebecomesevidentinelectrophoresisanalysisasatypicalladderpatternduetomultipleDNAfragments.However,althoughthisprotocolissimpleandgenerallyabletoprovidegoodresults,itisonlyqualitativebecauseofitslimitationsinDNArecoveryandsolubilization.InordertoobtainacleanerDNA,othermethodsforDNApreparationarerequired(insomecasesuseofproteinaseKfordeproteinizationisrecommended).

2.1.MaterialsCellsUSPensionat1-5x106cells/mlincompleteRPMImedium(A1)TTEsolution:TEbufferpH7.4(A1)with0.2%TritonX-100(storeat4°C)NaCl5M,icecoldIsopropanol,icecoldEthanolat70%,icecoldTEbufferpH7.4(A1)LoADIngbuffer10x(A1)TBEbufferforelectrophoresis(A1)Ethidiumbromidesolution(A1)Electrophoresis-gradeagaroseDNAmolecularweightMarkersRefrigeratedcellcentrifuge(A3)Microfuge(A3)Heatingblock(A3)Gelelectrophoresisapparatus(A3)DCpowersupply(A3)UVtransIlluminator(A3)PolaroidCamera+films(A3)

2.2.Methodology

1.Dispense0.5mlofcellsuspension(nolessthan5x105,otherwiseDNAwillnotbedetectablebyphotographyofethidiumbromidestainedgel,andnomorethan5x106,toavoiddifficulthandlingoftoohighamountsofinsolubleDNA)intubeslabeledB(bottom).

2.Centrifugecellsat200xgat4°Cfor10min.

3.TransfersupernatantscarefullyinnewtubeslabeledS(supernatant).

4.AddtothepelletintubesB0.5mlofTTEsolutionandvortexvigorously.Thisprocedureallowsthereleaseoffragmentedchromatinfromnuclei,aftercelllysis(duetothepresenceofTritonX-100intheTTEsolution)anddisruptionofthenuclearstructure(followingMg++chelationbyEDTAintheTTEsolution).

5.ToseparatefragmentedDNAfromintactchromatin,centrifugetubesBat20,000xgfor10minat4°C.

6.CarefullytransfersupernatantsinnewtubeslabeledT(top).

7.AddtothesmallpelletintubesB0.5mlofTTEsolution.

8.Addtothe0.5mlvolumepresentintubesB,SandT,0.1mlofice-cold5MNaClandvortexvigorously.TheadditionofthesaltshouldbeabletoremovehistonsfromDNA.

9.Addtoeachtube0.7mlofice-coldisopropanolandvortexvigorously.

10.Allowprecipitationtoproceedovernightat-20°C.Thisstepcanbeshortenedbyputtingsamplesinabathofethanol/dryicefor1hr.

11.Afterprecipitation,recoverDNAbypelletingfor10minat20,000xgat4°C.

12.Discardsupernatantsbyaspirationorbyrapidlyinvertingtubesandcarefullyremoveanydropsorfluidremainingadherenttothewallofthetubewithapapertowelcorner.Thiscanbeacriticalstepbecausethepelletcouldbeloosenandtransparent,hardtobeseen.

13.Rinsethepelletsbyaddingtoeachtube0.5-0.7mlofice-cold70%ethanol.

14.Centrifugetubesat20,000xgfor10minat4°C.

15.Discardsupernatantsbyaspirationorbyrapidlyinvertingtubes.Carefullyremoveanydropsorfluidremainingadherenttothewallofthetubebyinvertingtubesoveranabsorbentpapertowelfor30min.Letairdrythetubesinuprightpositionforatleast3hrbeforeproceeding.

16.DissolveDNAbyaddingtoeachtube20-50mlofTEsolutionandplacethetubesat37°Cfor1-3days.TheredissolutionofDNAmaybeacricalstep,infactitdependsontheDNAquantityandsizepresentinthesamples.Thus,thenon-fragmentedDNAcontainedintheBtubes,mayneedhighervolumesofTEandlongerincubationtimesinordertoberesuspended.

17.MixthesamplesofDNAwithloadingbufferbyadding10xloadingbuffertoafinalconcentrationof1x.Theadditionofloadingbuffertosamplesallowstoloadgelwellsmoreeasilyandtomonitortherunofsamples.

18.Placesamplesinaheatingblockat65°Cfor10minandimmediatelyload10-20mlofthemtoeachwellofastandard1%agarosegelcontainingethidiumbromide0.5mg/ml.AppropriateDNAmolecularweightmarkersshouldbeincluded.Ethidiumbromideisapotentialcarcinogen:wearglovesandhandlewithcare.

19.RuntheelectrophoresisinstandardTBEbufferaftersettingthevoltagetothedesiredlevel.DuringelectrophoresisitispossIBLetomonitorthemigrationofsamplesbyfollowingthemigrationofbromophenolbluedyecontainedintheloadingbuffer.

20.Stoptheelectrophoresiswhenthedyereachesabout3cmfromtheendofthegel.

21.TovisualizeDNA,placethegelonaUVtransilluminatorandtakephotosofthegel.WeareyeandskinprotectionwhenUVareon.

3.Commentary
3.1.Backgroundinformation

Apoptosisisaninnatemechanismofeukarioticcellsuicidewhichplaysamajorroleinmanyphysiologicalandpathologicalprocesses.Therefore,thedefinitionofcellularregulatorymechanismsandbiochemicalprocessesinvolvedinapoptosisisanimportantchallengefromboththeoreticalandappliedpointsofview.

Duringapoptosisaseriesofreorganisationoccurinthecell:chromatincondensation,lossofcellvolumeandmembraneblebbingaresomeofthemostevidentmorphologicalchangesofapoptoticcells.Althoughthemolecularmechanismsleadingtosuchchangesarenotcompletelyknown,manyofthemseemtoproceedinparallelwithbiochemicalevents.Thisisthecase,forexample,ofchromatincondensationandnuclearenvelopbreakdown.Infact,inparallelwiththemoccursDNAfragmentation,abiochemicalhallmarkofapoptosisinthemajorityofcells.ResponsibleforDNAcleavageisbelievedtobeanendogenousCa++-andMg++-dependentendonucleaseabletobreakdoublestrandDNAatinternucleosomalsites.Therefore,apoptoticDNAcleavageresultsincharacteristicfragmentsofoligonucleosomalsize(180-200bp).Suchphenomenum,describedforthefirsttimebyWyllie(1980),canbevisualizedbyanagarosegelelectrophoresisanalysis.ThepresentprotocolprovidesamethodforqualitativedeterminationofDNAfragmentation.

3.2.Criticalparameters

ThemostcriticalpointofDNAelectrophoreticalanalysisisitsinABIlityofquantitativemeasurementofapoptosis.Infact,duetoproblemslinkedtotheinsolubilityoflargeDNAandthustoitsfinalagarosegelanalysis,themethodisstrictlyqualitative.

Moreover,thismethodisnotrecommendedwhendifferentbehaviourinDNAfragmentationfollowingapoptoticstimuliisdescribed.Infact,insomecelltypeswhererandomdouble-strandedorraresingle-strandedDNAfragmentationoccur,itcannotbedetectedbyagarosegelelectrophoresisassay.

Sometimes,inparticularwithcellsobtainedfromexvivocultures(e.g.thymocytesandlymphocytes),anhighbackgroundofspontaneousDNAfragmentationcouldbeobserved.

3.3.Troubleshooting

Solubilizationofchromosome-lengthDNAcollectedintubesBisgenerallydifficult.IncreaseofTEvolumesandextensionofincubationtimemaybeneededfortheredissolutionofDNAfollowingprecipitation.Theuseoflimitednumberofcells(lessthan5x106)willbehelpfultolimitthisproblem.However,sincethemethodisexclusivelyqualitative,theanalysisoffragmentedDNApresentintubesTisthemaininterestoftheassay.

3.4.Anticipatedresults

TheassayofDNAagarosegelelectrophoresisprovidesgoodresultsforthedefinitionofcellapoptosis.AtypicalladderpatternofDNAfragmentationshouldbeobservedinmostapoptoticcells.
3.5.Timeconsiderations
PreparationofDNAforagarosegelelectrophoresisanalysisdependsontheDNAsizeofsamples:generallyfrom2to7daysarerequired.Additional4-8hrareneededforperformingelectrophoresis.
3.6.Keyreferences1.Wyllie,A.H.1980.Glucocorticoid-inducedthymocyteapoptosisisassociatedwithendogenousendonucleaseactivity.Nature284:555.

2.Duke,R.C.,andCohen,J.J.1986.Endogenousendonuclease-inducedDNAfragmentation:anearlyeventincell-mediatedcytolisis.Proc.Natl.Acad.Sci.U.S.A.80:6361.

3.Arends,M.J.,Morris,R.J.,andWyillie,A.H.1990.Apoptosis.Theroleoftheendonuclease.Am.J.Pathol.136:593.

4.Bortner,C.D.,Oldenburg,N.B.E.,andCidlowski,J.A.1995.TheroleofDNAfragmentationinapoptosis.TrendsCellBiol.5:21.

5.Sellins,K.S.,andCohen,J.J.1991.CytotoxicTlymphocytesinducedifferenttypesofDNAdamageintargetcellsofdifferentorigin.J.Immunol.147:795.

Appendix1(A1):Stocksolutions

Appendix2(A2):Reagents

RPMI-1640,500ml42402-016GibcoBRL

Gentamicinsulfate,solutionG-1522Sigma

L-glutamin20mM,200ml25030-024GibcoBRL

FCSA-1111-LHyclone

EDTAdisodiumsalt,dihydrateE-5134Sigma

TRIZMAbase(Tris)T-1503Sigma

TritonX-100115291ABioRad

SodiumChlorideS-9888Sigma

Isopropylalcohol412421CarloErba

Ethanol1170Riedel-deHaen

Laurylsulphate,sodiumsalt(SDS)L-4390Sigma

Ficoll400F-4375Sigma

BromophenolblueB-5525Sigma

XyleneCyanolX-4126Sigma

BoricacidB-0394Sigma

EthidiumbromideE-7637Sigma

DNAmolecularweightmarkersIX1449460BoehringerMannheim

Agarosestandard18054Eurobio

Polaroidfilmtype667F-4638Sigma

Appendix3(A3):Equipment

Multi-blockHeaterModel2094Lab-lineInstruments,Inc.

RefrigeratedcellcentrifugeModelGS-56RBeckman

RefrigeratedmicrocentrifugeModel5417REppendorf

VortexModelMT135CarloErba

WaterbathModel1002GFL

GelelectrophoresisapparatusModelHorizon58GibcoBRL

PowersupplyModel1000/500BioRad

UVTransilluminatorModelT2202Sigma

DirectscreeninstantcameraModelDS34Polaroid

Solution	Preparation	Storage
Complete RPMImedium	RPMI-1640mediumsupplementedwith5%heat-inactivatedfetalcalfserum(FCS),2mML-glutamine,25mMHEPESbuffer,50µg/mlgentamicinsulfate.L-glutamineislabile,thusitdoesnotlastat4°Cformorethanoneday.	4°C
TEbuffer	10mMTris.ClpH7.4(preparebydilutingstocksolution),1mMEDTA.	RT
Tris.Clstocksolution(1M)	Dissolve121gTrisbasein800mlH2O,adjusttodesiredpHwithconcentratedHCl,mixandaddH2Oto1liter. CAUTION:AdjustpHoftheTrisbufferatthesametemperatureatwhichitwillbeused,asthepHvarieswithtemperature(about0.028pHunitsper1°C).	RT
Loadingbuffer10x	Prepareconcentratedstocksolutionofloadingbufferbyaddingthefollowingreagentsattheindicatedfinalconcentrations:20%Ficoll400,0.1MEDTA(pH8.0),1%SDS,0.25%bromophenolblue,0.25%xylenecyanol(optional).	RT
TBEbufferstocksolution	Dissolvein800mlofH2O108gTrisbase(89mM),55gboricacid(89mM),40ml0.5MEDTA,pH8.0(2mM);bringto1literwithH2O.Usediluted1:10.	RT
Ethidiumbromidestocksolution	Dissolve50mgofethidiumbromidein100mlofH2O.Usediluted1:1000.	4°CProtectfromlight.
Agarosegel	Dissolve1%agarosein1xTBEbuffer(inthepresenceof0.5mg/mlethidiumbromide)byheatinguntilmelted.	Preparejustbeforeuse.