细胞结合实验

细胞结合实验细胞结合实验1|Preparecellsfortheexperiment.Thisprocedurewillpresentthedetailedcell-SELEXprotocolforbothsuspensionandadherentculturedcelllines.Foradherentcells,therearetwooptions:theexperimentcanbeperformedeitherdirectlyinaculturedishorusingdissociatedcells.Ifusingdissociatedcells,preparethembyfollowingthestepsgiveninBox1beforefollowingtheproceduregivenbelow.Ifperformingtheexperimentinaculturedish,refertoBox2andcontinuewiththeprocedurefromStep61.InitialDnalibrarypoolpreparation●tIMInG20min准备实验细胞。这个程序将详细的细胞SELEX协议和悬浮贴壁培养细胞系。贴壁细胞，有两种选择：实验可以直接在或使用解离的细胞培养皿。如果使用解离的细胞，他们准备通过下面1盒之前的步骤下面给出的程序如下。如果在培养皿中进行实验，涉及2盒并继续步骤61步骤。最初DNA文库池制备●定时20分钟2|Add20μlof0.5mM(10nmol)DNAlibraryto350μlofbindingbuffer,mixandheatthemixtureat95°Cfor5min.Snap-cooloniceandkeeponiceuntilreadytouse(sameday).pausepoIntIfselectionisnotaccomplishedonthesameday,storetheDNAlibrarypoolat?20°C.Thawlibraryonicewheneverreadytouse.ThedenaturationstepisnotnecessaryonceDNAhasbeenthawedonice.crItIcalstepHeatingtheDNAat95°CandsubsequentfastcoolingoniceareimportanttocreatefoldedssDNA.preparationoftargetcells:cellviability●tIMInG30min添加20μL0.5毫米（10nmol）350μL的结合缓冲液DNA文库，混合和热的混合物在95°C5分钟。卡酷冰保持在冰上待用（同一天）。暂停点如果选择不在同一天完成，在?20°C.解冻库在冰店DNA文库池当准备使用。变性步骤是不必要的一旦DNA已解冻的冰。关键步骤中加热到95°C和随后的快速冷却的冰上的DNA是创造折叠ssDNA重要。靶细胞的制备：细胞活力●定时30分钟3|DeterminecellviabilityusingTrypanblueexclusionassay.crItIcalstepCellviabilityassessmentisveryimportant,especiallyforsuspensioncells.Toomanydeadcellswillseri-ouslyaffecttheefficiencyofselection.DNAcannonspecificallyadheretoandenterdeadcells.Thiswillcausethelossofimportantsequences,delayofenrichmentoreventhefailureofselection.Asthehighestcellviabilityisideal,itispreferabletousemorethan95%viablecells.preparationoftargetcells:cellnumber●tIMInG20min4|Determinetheconcentrationofcellsusingahemocytometer.Onthebasisofcellcount,determinewhatvolumewillcorrespondtothenumberofcellsneededforaspecifcroundofselection.crItIcalstepForthefirstroundofselection,usethehighestnumberofcellspossibleforcollectingspecificsequences,preferablybetween5and10millioncells.preparationoftargetcells:washingbycentrifugation●tIMInG30min5|Takethecellvolumethatyieldsthedesirednumberofcellsintoa15-mlcentrifugetubeandcentrifugecellsat150gfor3minat4°C.Removethesupernatantandadd3mlofwashingbuffer.Resuspendcellsbypipettingupanddown,tappingthebottomofthecentrifugetubeormildvortexing.Pelletthecellsatthesamespeedandduration.Repeatwashingoncemore.crItIcalstepAvoidstrongvortexingasitcancausecellbreakageandmayeventuallyaffectselection.IncubationofcellswithDnalibrarypool●tIMInG1h6|Resuspendcellsin330μlofbindingbuffer.Addallofthe370μlsnap-cooledDNAlibrary(Step2)tothe330μlcellsuspension,mixthoroughlyandincubatethemixtureonicefor1honarotaryshaker.IncubationtemperaturedependsonBoX1|DISSoCIATEADHERENTCELLS●tIMInG4H15mIN1.Splitcells24hbeforedissociationtreatment.2.Dissociatecellsfromculturefaskordishwithnonenzymaticdissociationsolutionorbyshort-term(30s–1min)trypsintreatment.Afterincubation,removetrypsinordissociationbufferandimmediatelyaddcoldculturemediumcontainingFBS.Cellsaresplit24hbeforedissociation;hence,theycanberemovedbyshort-termtreatment.Ifcellsareleftfor2–3d,thiswillnotbepossible.3.Transfercellsfromfaskordishtocentrifugetubeforsubsequentuse.FBSwillhaltfurtheractionofresidualtrypsin.4.Forcellsdissociatedwithnonenzymaticdissociationbuffer,countcellsanduserequiredamountforaptamerselectionbyfollowingthesameprocedureasdescribedforsuspensioncells(Steps2–60).5.Forcellsdissociatedbyshort-termtrypsintreatment,usecellsimmediately,asdescribed(Steps2–60),orincubatecellsundercellcultureconditions,butwithrockingforatleast2h.crItIcalstepContinuousrockingwillpreventthecellsfromstickingtothebottomofthefaskordish.Itisimportanttoculturecellsforatleast2htorecoversomeoftheproteinsthatmighthavebeenaffectedbytheshort-termtrypsintreatment.6.Afterincubation,countthenumberofcellsandusethemforselection,followingthesameprocedureasdescribedforsuspensioncells(Steps2–60).5.Forcellsdissociatedbyshort-termtrypsintreatment,usecellsimmediately,asdescribed(Steps2–60),orincubatecellsundercellcultureconditions,butwithrockingforatleast2h.crItIcalstepContinuousrockingwillpreventthecellsfromstickingtothebottomofthefaskordish.Itisimportanttoculturecellsforatleast2htorecoversomeoftheproteinsthatmighthavebeenaffectedbytheshort-termtrypsintreatment.6.Afterincubation,countthenumberofcellsandusethemforselection,followingthesameprocedureasdescribedforsuspensioncells(Steps2–60).crItIcalstepTrypsintreatmentmustbeperformedwithextremecautionbecausesubstantialdamagetosurfaceproteinscanoccurifthetreatmentisprolonged.Moreover,donottreatcellswithprolongedexposuretotheharshnonenzymaticdissociationbuffer.Atthemost,5minissuffcient.Bothhigherconcentrationandlong-termexposurearedetrimentaltocellintegrityandsurfacemarkers.trouBlesHootInGBoX2|PERFoRmSELECTIoNDIRECTLYINCULTUREDISH●tIMInG1H40mIN1.Culturecellsintomonolayerswithatleast90%confuence.crItIcalstepPerformingselectiondirectlyintheculturedishmaintainscellsintheirnaturalenvironmentandproteinsintheirnativeconformation.Thereislessconcernofcellsurfacevariability.2.Forthefrstroundofselection,usea100-mm×20-mmculturedish(cellnumberover5million).Washcellswithwashingbuffertwice.crItIcalstepDonotuseaculturefaskwhenperformingselectiondirectlywithadherentmonolayerbecauseitiseasiertowashandremovethecellsfromanuncovereddishthanfromafask.3.PrepareDNAlibraryin500μlofbindingbuffer.4.IncubatecellswithDNAlibrary.Useatotalvolumeof1,000μlforthe100-mm×20-mmculturedishandshakecontinuously(50r.p.m.)oronslowrockerfor1h.crItIcalstepBecausetheDNAlibraryvolumeisexpectedtocovertheentireculturedish,adjustthetotalvolumeoftheDNAlibraryto1,000μlwhilemaintainingthefnalconcentration.5.Afterincubation,washcellsthreetimeswithwashingbuffer.crItIcalstepEnsurethatthecellsarenotdetachedduringwashing,asthiswillcauselossofbindingsequences.6.Add500μlofDNAse-freewatertothewashedcells.Detachcellsusingcellscraperandtransferthemintoa1.5-mltube.7.Heatsuspensionat95°Cfor10minandrecoverthesuspendedDNAbycentrifugationat13,100gfor5min.Ensurethattherecoveryeffciencyisashighaspossible.8.FollowSteps10–34toobtainthefrstselectedDNApool.pausepoIntOnemaystoretheDNApoolat?20°C.secondroundofselection9.Usethegeneratedfrst-roundselectedpool,resuspendedinbindingbuffer.10.FollowBox2,steps1–5.11.Elutethecell-DNAcomplexin600μlofbindingbuffer.Detachcellsusingcellscraperandtransferthemintoa1.5-mltube.HeatandrecovertheelutedDNAsuspensionbycentrifugation.negativeselection12.Usea100-mm×20-mmculturedishwithcellsinconfuence.crItIcalstepAvoidnonconfuentcellcultureasDNAcansticktothebareculturedish.13.Washthecells.14.Incubatecellswiththeelutedpool.15.Afterincubation,recoverthebuffercontainingtheremainingsequencesthatdonotbindtothecontrolcells.LabelthisasthesecondselectedDNApool.pausepoIntOnemaystoretheselectedDNApoolat?20°C.pcrprocedures—ssDna16.FollowSteps15–34toobtainssDNAsubsequentroundsofselection17.Usea60-mm×15-mmculturedishtocarryouttheincubationstepusing250μlofthessDNApoolwith250μlofbindingbufferandmaintaininga1,000nMfnalconcentrationoftheselectedDNAlibrary.Followwiththewashingandelutionsteps.pausepoIntOnemaystoretheselectedDNApoolat?20°C.Monitorprogressofselection18.Onemaydissociatecellsandusesimilarbindingassaysasdescribedforsuspensioncells(Steps52–59)oruseconfocalmicroscopywhenadherent.1174|VOL.5NO.6|2010|natureprotocolsthepurposeofselection.Ingeneral,anytemperaturebetween4and37°Cisapplicableforthisprotocol;however,highertemperaturessuchas37°Ccancauseinternalization.Onceaptamerisselectedat4°C,itisimportanttotestthebindingat37°C.Thisdoesnotmeanthatallaptamersselectedat4°Cwillnotbindat37°C.Fromobservation,mostoftheaptamerswillbindverywellat37°C,especiallythosewithveryhighaffnity.Someoftheaptamersgeneratedat4°Chavebeenusedinvariousapplicationsat37°C(refs.30,33).Themostimportantpointhereistogenerateaptamerswithhighaffnitybygraduallyincreasingthestringencyofselectiontogenerateaptamersthatcanbindatdiversebindingconditions.Forinstance,aptamersthathavebeendevelopedusingDPBSasthebindingbuffercanstillrecognizethetargetinculturemedium.crItIcalstepBecauseofthelargenumberofcellsrelativetothesmallvolumeofincubationmedium,itissometimescommontoseecellssettlingatthebottomofthetube,evenwhileshaking,sooccasionallycheckandresuspendthecells.Washingstep●tIMInG40min7|Afterincubation,centrifugecellsat150gfor3minat4°C.Removesupernatantcontainingunboundsequencesandresuspendcellpelletsin3mlofwashingbuffer.Shakeforabout30sandcentrifugeagainusingthesamecondition.Removesupernatantcarefullywithtransferpipette,avoidingcellloss.Quicklyspindown(30s)theresidualbufferonthewallofthetube.Toavoidcellloss,removeasmallamountofresidualbufferwithfoldedkimwipe.Repeatthewashingproceduretwomoretimesforatotalofthreewashings.trouBlesHootInGelutionofboundsequences●tIMInG15min8|Forthefrstroundofselection,elutetheboundsequencesinwater.Add500μlofDNase-freewatertothecellpellet.Resuspendcellsandtransfercellsuspensionintoa1.5-mlmicrofugetube.crItIcalstepOnlyinthefirstroundisDNAelutedinwater.Insubsequentrounds,DNAiselutedinbindingbuffer.AstheentireelutedpoolisamplifiedbyPCR,useonlyDNase-freewaterfortheelutionoftheboundDNAsequencesduringthefirstroundofselection.Otherwise,iftheDNAiselutedinDPBS,thesaltswithhighconcentrationwillaffecttheefficiencyofPCR.9|Heatthecellmixtureat95°Cfor10min,centrifugeat13,100gfor5minandcollectsupernatantcontainingelutedDNA.crItIcalstepDonotperformnegativeselectionatthispoint.Minimizethelossofelutedsequencesduringthefirstselectionround,aseachsequenceistheoreticallyrepresentedonlyonce,andwhenanysequenceislost,itcanneverberecovered.pausepoIntStoreelutedDNAat?20°C.pcrprocedures:pcramplificationoftheentirefirstselectedpool●tIMInG1h10|Thisstepisperformedforpoolsgeneratedfromonlythefrstselection.Setupa1,000-μlPCRamplifcationreactionvolumeasshownbelow:adjustthevolumeofwatertocompensateforthetotalvolumeiftheDNAselectedpoolvolumediffers.reagentsreactionmixture(μl)10×PCRbuffer100.0dNTPmixture(2.5mMeach)80.0FITC-andbiotin-primermixture50.0(0.5μMfinal)DNAselectedpool(template)500.0DNase-freewater270.0HSTaqpolymerase3.011|Mixthoroughlyandpipet100μlinto10individualtubes(using96-wellthermalcycler)for100μlperreaction,orpipet200μlintofveindividualtubes(60-wellthermalcycler)for200μlperreaction.crItIcalstepNotethatthisstepisonlyperformedtostepupthecopiesofindividualsequencesandisnotnecessar-ilypreparative.Toomanycyclesmayproducenonspecificamplicons,andthiswillaffectthepurityoftheDNAlibrarypool.Choose8–10cycles.12|PerformPCRamplifcation.Forexample,inprimerset2givenabove,usethefollowingamplifcationconditions:denaturationat95°Cfor30s,annealingat56.3°Cfor30sandelongationat72°Cfor30s,followedbyfnalextensionfor3minat72°C.crItIcalstepAlwayskeepTaqpolymeraseat?20°Cuntilreadytouse.ThawandkeepallotherPCRreagentsonice.steptemperature(°c)time(s)Hotstart95150Amplification(10cycles)Denaturation9530Annealing56.330Extension7230Finalextension72180Hold413|AfterPCRamplifcation,poolallreactionmixturestogether(total1,000μl).Thereisnoneedtoperformagarosegelelectrophoresistoassesstemplateamplifcation.Inmostcases,thefrst-roundPCRamplifcationproductisinsuffcientfordetectionbyethidiumbromide.crItIcalstepNotethatthePCR-amplifiedpoolcanpotentiallycontaminatePCRreagentsandprimers;therefore,keepthemseparated.14|LabelthePCRproductasthefrstselectedpool.pausepoIntStoretheDNApoolat?20°C.pcrprocedures:determinetheoptimumnumberofcyclesforpreparativepcr●tIMInG1h30min15|ThetotalPCRreactionmixturevolumeforeachtubeis50μl,withtheamplifedfrstselectedpoolat10%servingasthetemplate.ChoosePCRsamplesatthefollowingcycles:4,6,8,10and12,andonenegativecontrolatthetwelfthcycle.AddthefollowingreagentstoPCRtubesasshownbelow:reagentspositivereactionmixture(μl)control(μl)10×PCRbuffer25.05.0dNTPmixture(2.5mMeach)20.04.0FITC-andbiotin-primermixture12.5(0.5μMfinal)2.5AmplifiedDNAselectedpool(template)25.0(10%ofreactionmixture)—Supernatantofpurecelllysate—5.0DNase-freewater166.7533.35HSTaqpolymerase0.750.15Abbreviation:FITC,fuoresceinisothiocyanate.16|Mixthoroughlyandpipet50μlofthepositivereactionmixtureintofveindividualtubes.17|PerformPCRamplifcationusingthePCRprogramdescribedinStep12withthemaximumnumberofcyclesas12.18|OpenthethermalcyclerandtakesamplesatthespecifedcyclesasshowninStep15.pcrprocedures:agarosegelelectrophoresis●tIMInG1h40min19|Prepare3%agarosegel.20|Prepareagarosegelsamplesasshownbelow:positive(μl)control(μl)ladder(μl)Blue/greenloadingdye(6×)222PCRproduct10——PCRnegativecontrol—10—Ladder(25bp)——1H2O——921|Loadsamplesinlanesandperformelectrophoresisat100Vfor40min.22|Removethegel,stainwithethidiumbromide,andobservethebandsunderUVlightandtakeimageofthegel(Fig.2).trouBlesHootInG23|Selectacyclenumberthatyieldsabrightbandwithoutnonspecifcamplicons.OnthebasisofFigure2,agoodexampleisthetenthcycle(lane5).InpreparativePCR,usethiscycleofamplifcationtoproducemorePCRproductsfortheprepara-tionofssDNArequiredforthesecondroundofselection.pausepoIntOnemaystorethelibrarypoolat?20°C.pcrprocedures:preparativepcr●tIMInG2h24|UsethesamereagentconcentrationsaslistedinStep15,butinlargervolumes.Forthesecondroundofselection,1,000μloftotalPCRreactionmixtureisenoughtogeneratetherequiredamountofssDNA.Moreover,theentiressDNApoolthatisgeneratedhereisusedforselec-tion,whereasinthehigherrounds,someareusedformonitoringtheselectionprogress.PreparePCRreactionmixtureasshownbelow:reagentspositivereactionmixture(μl)10×PCRbuffer100.0dNTPmixture(2.5mMeach)80.0FITC-andbiotin-primermixture50.0(0.5μMfinal)AmplifiedDNAselectedpool(template)100.0(10%ofreactionmixture)DNase-freewater670.0HSTaqpolymerase3.0Abbreviation:FITC,fuoresceinisothiocyanate.1234567Figure2|AgarosegelelectrophoresisimageshowingtheproductsofthevariouscyclesofselectedDNAlibraryamplification.Lane1=25-bpladder;lane2=4cycles;lane3=6cycles;lane4=8cycles;lane5=10cycles;lane6=12cycles;andlane7=negativecontrol.crItIcalstepItisnotnecessarytoperformcontrolPCRforthepreparativeprocessoncethecycleoptimizationcontroldataareclean.25|Mixthoroughlyandpipet100μlor200μlofthemixtureintotenPCRtubes(96-wellthermalcycler)orfvePCRtubes(60-wellthermalcycler).ClosethecapsecurelyandperformPCRusingtheprogramdescribedinStep12,butwiththechosennumberofcycles.26|AfterPCR,poolallPCRproductstogetherandperformgelelectrophoresis(Step20)toconfrmthePCRproduct(Fig.3).pausepoIntOnemaystorethelibrarypoolat?20°C.preparationofssDnafrompcrproduct●tIMInG40min27|InsertaflterandsecureitfrmlyatoneendofanemptyDNAsynthesiscolumn.Removetheplungerfroma10-mlsyringewithoutaneedleandinserttheemptysyringeattheotherendoftheDNAsynthesiscolumn.28|Mountthesetuponaclamp.Add200μlofstreptavidinSepharosebeadssuspension.Inserttheplungerandallowthestoragebuffertodrain.29|Washthebeadswith2.5mlof1×DPBS.30|PassthePCRproductthreetimesthroughthecolumn.Washthebeadsagainwith2.5mlofDPBS.31|Add500μlof200mMNaOH,inserttheplungerandcollecttheeluatecontainingtheFITC-labeledssDNA.crItIcalstepAllowthePCRproductandtheNaOHsolutiontopassthroughthecolumngradually.PushingtheplungerhardwillreducetheeffciencyofthessDNAyield.Furthermore,donotremovetheplungerfromthesyringe21Figure3|AgaroseelectrophoresisgelimageofpreparativeselectedDNAlibrarypool,confirmingtheefficiencyofamplification.Lane1=25-bpladderandlane2=PCRproductof10cycles.whilethesyringeisconnectedtothecolumncontainingthebeads.Alwaysdisconnectthesyringefromthecolumnbeforeremovingtheplunger.trouBlesHootInGDesaltingssDna●tIMInG30min32|WashtheNAP5columnwithatleast15mlofdeionizedwater.Add500μlofelutedssDNA.AllowtheentiresamplevolumetodrainandthenelutethessDNAwith1,000μlofDNase-freewater.crItIcalstepAlwayskeepthesurfaceofthedesaltingcolumnmoist.trouBlesHootInGQuantifyingelutedssDna●tIMInG5min33|DeterminetheconcentrationofssDNAbyUVabsorbanceat260nm.DryingssDna●tIMInG3h34|ConcentratethessDNAusingaDNASpeedvacdryer.Technically,thisstepistheendofoneroundofDNAaptamerselection.pausepoIntOnemaystorethedriedssDNAat?20°Candresuspenditinbindingbufferwhenneededforthenextroundofselection.trouBlesHootInGsecondroundofselection●tIMInG2h30min35|ResuspendssDNAinbindingbuffertoobtainaconcentrationof1,000nM.36|DenatureandrenaturethessDNApoolasdescribedinStep2.preparationofcellsforsecondroundofselection37|FollowthesameprocedureusedinSteps3and4butwithlessthan5millioncells.Washing38|RepeatStep5.Dnaincubation39|Add200μlofssDNA(Step36)to200μlofcellsuspension.RepeatSteps7,8(use600μlbindingbuffer)and9.pausepoIntOnemaystoretheselectedDNApoolat?20°C.negativeselectionprocedurecellviabilityandnumberofsuspensioncells●tIMInG2h30min40|CheckcellviabilityandcountcellsasdescribedinSteps3and4.Useatleast10millioncontrolcells.crItIcalstepTheviabilityofcontrolcellsisimportantinnegativeselectionbecausesequencesthatnonspecifcallyadheretoandenterdeadcellswillcausethelossofspecifcsequences.Totheextentpossible,usemorethan95%viablecells.41|WashcellsasdescribedinStep5.42|IncubatecellpelletwiththeentireelutedDNApoolfrompositiveselectiononicewithcontinuousshakingfor1h.43|Afterincubation,spindownthecellscontainingthebindingsequencesat3,059gfor5min.44|RecoverthesupernatantcontainingsequencesthatdonotbindtocontrolcellsandlabelthispoolasthesecondselectedDNApool.pausepoIntOnemaystoretheselectedDNApoolat?20°C.pcramplifcationsDeterminationoftheoptimumnumberofcyclesforpreparativepcrofthesecondselectedDnapool●tIMInG3h10min45|EstimatetheminimumandmaximumnumberofcyclestobeassessedonthebasisofthepreviousPCRcycleoptimizationprocedures(Steps15–23),butusethesecondselectedDNApoolasthetemplate.Rtocolpreparativepcr●tIMInG2h46|FollowthesameprocedureasdescribedforSteps24–26(Fig.4,panelb).pausepoIntOnemaydrytheselectedDNApoolandstoreitat?20°C.ssDnapreparation●tIMInG40min47|FollowSteps27–31.Desalting,quantifyinganddrying●tIMInG3h35min48|FollowsSteps32–34.subsequentroundsofDnaaptamerselection●tIMIn