IntroductionInourlabweusetheterm\"cell-freesystem\"whenwetalkabouttheexaminationofapoptoticactivityincytoplasmicextracts.Thecytoplasmicextractsmaybepreparedfromcellswhichweretreatedinculturewithanapoptosis-inducingagentorfromuntreatedcells.Inthelattercase,apoptoticactivitycanbeinducedbyadditionofapoptoticstimuli(suchasrecombinantactivecaspasesorcytochromec)totheextracts.Apoptoticactivityinthecytoplasmicextractscanbeexaminedbythemeasurementofenzymaticcaspase-acticity,bywesternblotsofproteinsprocessedduringapoptosis(caspasesandtheirsubstrates),orbyusingisolatedcellnucleiasindicatorsforapoptoticfactorsinducingnuclearmorphologicalchangesand/orDNAfragmentation.Itwasin1993whenthefirstpaperdescribedthatacell-freesystemcanmimiccharacteristicfeaturesofapoptosisinintactcells(Lazebniketal.,1993,J.CellBiol.,123(1):7-22).Indeed,ithasbecomeclearthattheapoptoticpathwaysactinginthecytoplasmfunctionindependentlyfromthenucleusandthus,cell-freesystemsappeartobeappropriatemodelsystemswhichrepresentatleastpartoftheapoptoticmachineryandsignalingmechanisms.Cell-freesystemshavebeenpreviouslysuccessfullyappliedinthedissectionofbiochemicalmechanismsduringtheapoptoticprocess,suchastheidentificationandcharacterizationofthe\"apoptosome\",AIF,andtheDNAfragmentationfactorICAD(Zouetal.,1997,Cell,90:405-413;Susinetal.,1999,Nature,397:441-446;Enarietal.,1998,Nature,391:43-50).Alsosignalingpathwayssuchasthecaspase-cascadehavebeenstudiedundercell-freeconditions,povidinginsightintoactivationpatternsandinhibitor-specificities(Mesneretal.,1999,JBC,27422635-22645;Faleiroetal.,1997,EMBO,16(9):2271-2281;Takahashietal.,1997,Oncogene,14:2741-2752).TheprotocolsdescribedbelowarederivedessentiallyfromLazebniketal.,1993,J.CellBiol.,123(1):7-22andFearnheadetal.,1997,GenesandDev.,11:126-1276. Activationofapoptoticactivityincytoplasmicextractsandmeasurementoftheresultingcaspaseenzymaticacticity Cytoplasmicextractswereactivatedaccordingtofollowingprotocol(asanexample,cytochromec/dATPareusedasapoptoticstimuli).Thecomponentsweremixedinthegivenorderandthenincubatedat37°Cusuallyfor45min. xµlDilutionBuffer(DB)containingATPregenerationsystem+1.5µlcytochromecfrombovineheart(50µM)+1.5µldATP(10mM)+yµlcytoplasmicextract(37.5µgprotein) xwassoadjustedthatthetotalvolumewas15µlandthusthefinalproteinconcentrationat2.5µg/µl. Whenactiverecombinantcaspases(suchascaspase-8)areusedforactivationoftheextracts,100ngofrecombinantcaspaseisaddedinsteadofcytoc/dATP. Incasethattheeffectofinhibitorswasstudied,suchasAc-DEVD-fmk,orzVAD-fmk,theinhibitoratconcentrationsrangingfrom100nMupto100µMwaspreincubatedtogetherwiththeextractinDBfor5min,thencytoc/dATPwasadded. Afterincubationoftheextractsinthepresenceorabsenceofactivatingstimuli(cytochromec/dATPoractiverecombinantcaspase),caspaseenzymaticactivitywasmeasuredaccordingtofollowingprotocol: 200µlofCaspaseAssayBuffer(CAB)containing20µMfluorescentsubstrate(Ac-DEVD-amcAc-YVAD-amc,Ac-VEID-amc;Calbiochem)wasaddedtotheactivationreaction.Thesamplesweretransferedintothewellsofa96flat-bottomwellplate.Afterincubationinthedarkfor30minatRT,fluorescencewasmeasuredat360/460nmwithaFL500fluorimeter(Bio-Tek).Ifastandard-curveofvariousconcentrations(0upto5µMinCAB)ofaminomethylcoumarin(amc)ismeasured,theenzymeactivitycanbecalculatedandexpressedaspikomolesofsubstrate(amc)hydrolyzedpermicrogramofprotein(intheextract)perminute[pmol•µg-1•min-1]orjustaschangeoftheamcconcentrationinnanomolarpermicrogamofprotein[nM•µg-1]. Activationofapoptoticactivityincytoplasmicextractsforwesternblotanalysis Cytoplasmicextractswereactivatedaccordingtofollowingprotocol(asanexample,cytochromec/dATPareusedasapoptoticstimuli).Thecomponentsweremixedinthegivenorderandthenincubatedat37°Cusuallyfor1h. xµlDilutionBuffer(DB)containingATPregenerationsystem+0.46µlcytochromecfrombovineheart(325µM)+0.3µldATP(100mM)+yµlcytoplasmicextract(75µgprotein) xwassoadjustedthatthetotalvolumewas30µlandthusthefinalproteinconcentrationat2.5µg/µl. Whenactiverecombinantcaspases(suchascaspase-8)areusedforactivationoftheextracts,100ngofrecombinantcaspaseisaddedinsteadofcytoc/dATP. Incasethattheeffectofinhibitorswasstudied,suchasAc-DEVD-fmk,orzVAD-fmk,theinhibitoratconcentrationsrangingfrom100nMupto100µMwaspreincubatedtogetherwiththeextractinDBfor5min,thencytoc/dATPwasadded. Afterincubationoftheextractsinthepresenceorabsenceofactivatingstimuli(cytochromec/dATPoractiverecombinantcaspase),westernblotswereperformedloADIngusually20µgproteininsamplebufferperlaneona4-20%SDS-PAGEgel. Reconstitutionofactivatedcytoplasmicextractswithisolatednuclei.AnalysisofapoptoticactivitybyqualitativeDNAladderingassayandDAPIstaining. CytoplasmicextractsweredilutedwithDBtoaconcentrationof7.5µgproteinpermlinatotalvolumeof50µl.Theextractswereactivatedornotactivatedbyadditionofe.g.10µMcytochromecand1mMdATP,then300,000isolatednucleiinavolumeof1.5µlNSB(e.g.fromJurkatcells)wereadded.Thereactionswereincubatedat37°Cfor4h. Hereisanexampleforareactionmixforthiskindofexperiment: ycorrespondsto375µgprotein,andxiscalculatedsothatthefinalvolumeis50µl. Afterincubation,thenucleiwereeitheranalyzedbyDNAladderingorDAPIstainingorboth,asdescribedbelow. Reconstitutionofactivatedcytoplasmicextractswithisolatedradioactivenuclei.Analysisofapoptoticactivitybyquantitative,radioactiveDNAfragmentationassaysorDAPIstaining. RadioactiveDNAfragmentationassay Radioactivenuclei(e.gfromALVA31cells)werepreparedasdescribedintheprotocol\"Isolationofcellnucleifortheapplicationinacell-freesystem\".Priortouseinthecell-freesystem,5x104nucleiinNSB(5µlof107nucleiperml)weredistributedin0.5mlmicrofugetubesandwerewashedoncein50µlDB.Thenucleiwerethenincubatedincytoplasmicextracts(finalproteinconcentration7.5mg/ml)inthepresenceorabsenceof10µMcytcand1mMdATPinatotalvolumeof10µlfor4hat37°C(650nuclei/µgprotein). Hereisanexampleforareactionmixforthiskindofexperiment: ycorrespondsto75µgprotein,andxiscalculatedsothatthefinalvolumeis10µl. Afterincubation,thenucleiweretransferredfromthemicrofugetubesintothewellsofa96wellplate;thenuclei\"sDNAwasharvestedonaglassfibermembraneandtheretentedradioactivitymeasuredbyscintillationcounting.Experimentswererunintriplicateorpentuplicateforeachcondition.ThepercentageofDNAfragmentationwascalculatedasfollows: ([cpmofnucleiinpureextracts]-[cpmofnucleiinextracts+cytoc/dATP])/[cpmofnucleiinpureextracts]x100 MATERIAL ExtractDilutionBuffer(DB): COMPOSITION: RECIPEfor500µl: 10mMHEPES(pH7.0)5mMEGTA50mMNaCl2mMMgCl21mMDTT supplementedwithATPregenerationsystem: 2mMATP10mMphosphocreatine50µg/mlcreatinekinase 100µlof50mMHEPES,pH7.0incl.25mMEGTA25µlof1MKCl10µlof100mMMgCl20.5µlof1MDTT - 5µlof200mMATPinwater10µlof500mMphosphocreatineinwater10µlof2.5mg/mlcreatinekinaseinKPMbuffer 365µlH2Onucleasefree AddDTTandATPregenerationsystemalwaysfreshtothebuffer,justbeforeuse! CaspaseAssayBuffer(CAB): COMPOSITION: RECIPEfor50ml: 50mMPIPES0.1mMEDTA10%glycerol1mMDTT 838.4mgPIPES10µlof0.5MEDTA5mlglycerol- add40mlH2O,adjustpH=7.2usingKOH(about200µlof1MKOH),thenfilluptothefinalvolumeof50ml. 1mMDTTisalwaysaddedfreshtothebuffer,justbeforeuse. LysisBuffer(forDNAisolationfromnuclei) COMPOSITION: RECIPEfor50ml: 50mMTris-HCl,pH8.010mMEDTA0.2%SDS 5mlof0.5MTris-HCl,pH8.21mlof0.5MEDTA,pH8.01ml10%SDS add43mlH2O TEBuffer COMPOSITION: RECIPEfor50ml: 50mMTris-HCl,pH8.01mMEDTA 5mlof0.5MTris-HCl,pH8.2100µlof0.5MEDTA,pH8.0 add44.9mlH2O 4%ParaformaldehydeinPBS RECIPEfor100ml: Solutionisgoodforatleast1year.Workunderahood!!!
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