细胞结合实验

细胞结合实验

1|Preparecellsfortheexperiment.Thisprocedurewillpresentthedetailed

cell-SELEXprotocolforbothsuspensionand

adherentculturedcelllines.Foradherentcells,therearetwooptions:the

experimentcanbeperformedeitherdirectlyina

culturedishorusingdissociatedcells.Ifusingdissociatedcells,preparethem

byfollowingthestepsgiveninBox1before

followingtheproceduregivenbelow.Ifperformingtheexperimentinaculture

dish,refertoBox2andcontinuewiththe

procedurefromStep61.

InitialDnalibrarypoolpreparation•tIMInG20min

准备实验细胞。这个程序将详细的细胞九LD（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|Add20plof0.5mM(10nmol)DNAlibraryto350plofbindingbuffer,mix

andheatthemixtureat95°Cfor5min.

Snap-cooloniceandkeeponiceuntilreadytouse(sameday).

pausepointIfselectionisnotaccomplishedonthesameday,storetheDNA

librarypoolat?20℃.Thawlibraryonice

wheneverreadytouse.ThedenaturationstepisnotnecessaryonceDNAhas

beenthawedonice.

criticalstepHeatingtheDNAat95℃andsubsequentfastcoolingoniceare

importanttocreatefoldedssDNA.

preparationoftargetcells:cellviability•tIMInG30min

添加20pL0.5毫米(10nmol)350|JL的结合缓冲液DNA文库，混合和热的混合物

在95℃5分钟。

卡酷冰保持在冰上待用(同一天)O

暂停点如果选择不在同一天完成，在?2CTC.解冻库在冰店DNA文库池

当准备使用。变性步骤杲不必要的一日DNA已解冻的冰。

关键步骤中加热到95℃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,especiallyfor

suspension

cells.Toomanydeadcellswillseri­

ouslyaffecttheefficiencyofselection.DNAcannonspecificallyadheretoand

enterdeadcells.Thiswillcausethelossof

importantsequences,delayofenrichmentoreventhefailureofselection.As

thehighestcellviabilityisideal,itispreferable

tousemorethan95%viablecells.

preparationoftargetcells:cellnumber•tIMInG2Cmin

4|Determinetheconcentrationofcellsusingahemocytometer.Onthebasis

ofcellcount,determinewhatvolumewill

correspondtothenumberofcellsneededforaspecifcroundofselection.

criticalstepForthefirstroundofselection,usethehighestnumberofcells

possibleforcollectingspecificsequences,

preferablybetween5and10millioncells.

preparationoftargetcells:washingbycentrifugation•UMInG30min

5|Takethecellvolumethatyieldsthedesirednumberofcellsintoa15-ml

centrifugetubeandcentrifugecellsat150g

for3minat4℃.Removethesupernatantandadd3mlofwashingbuffer.

Resuspendcellsbypipettingupanddown,

tappingthebottomofthecentrifugetubeormildvortexing.Pelletthecellsat

thesamespeedandduration.Repeat

washingoncemore.

criticalstepAvoidstrongvortexingasitcancausecellbreakageandmay

eventuallyaffectselection.

IncubationofcellswithDnalibrarypool•tIMInG1h

6|Resuspendcellsin330plofbindingbuffer.Addallofthe370plsnap-cooled

DNAlibrary(Step2)tothe330plcell

suspension,mixthoroughlyandincubatethemixtureonicefor1honarotary

shaker.Incubationtemperaturedependson

BoX1|DISSOCIATEADHERENTCELLS•tIMInG4H15mlN

1.Splitcells24hbeforedissociationtreatment.

2.Dissociatecellsfromculturefaskordishwithnonenzymaticdissociation

solutionorbyshort-term(30s-1min)trypsintreatment.

Afterincubation,removetrypsinordissociationbufferandimmediatelyadd

coldculturemediumcontainingFBS.Cellsaresplit24h

beforedissociation;hence,theycanberemovedbyshort-termtreatment.If

cellsareleftfor2-3d,thiswillnotbepossible.

3.Transfercellsfromfaskordishtocentrifugetubeforsubsequentuse.FBS

willhaltfurtheractionofresidualtrypsin.

4.Forcellsdissociatedwithnonenzymaticdissociationbuffer,countcellsand

userequiredamountforaptamerselectionbyfollowing

thesameprocedureasdescribedforsuspensioncells(Steps2-60).

5.Forcellsdissociatedbyshort-termtrypsintreatmentusecellsimmediately,

asdescribed(Steps2-60),orincubatecellsundercell

cultureconditions,butwithrockingforatleast2h.

criticalstepContinuousrockingwillpreventthecellsfromstickingtothe

bottomofthefaskordish.Itisimportanttoculture

cellsforatleast2htorecoversomeoftheproteinsthatmighthavebeen

affected

bytheshort-termtrypsintreatment.

6.Afterincubation,countthenumberofcellsardusethemforselection,

followingthesameprocedureasdescribedforsuspension

cells(Steps2-60).

5.Forcellsdissociatedbyshort-termtrypsintreatment,usecellsimmediately,

asdescribed(Steps2-60),orincubatecellsundercell

cultureconditions,butwithrockingforatleast2h.

criticalstepContinuousrockingwillpreventthecellsfromstickingtothe

bottomofthefaskordish.Itisimportanttoculture

cellsforatleast2htcrecoversomeoftheproteinsthatmighthavebeen

affectedbytheshort-termtrypsintreatment.

6.Afterincubation,countthenumberofcellsardusethemforselection,

followingthesameprocedureasdescribedforsuspension

cells(Steps2-60).

criticalstepTrypsintreatmentmustbeperforrredwithextremecaution

becausesubstantialdamagetosurfaceproteinscanoccur

ifthetreatmentisprolonged.Moreover;donottreatcellswithprolonged

exposuretotheharshnonenzymaticdissociationbuffer.At

themost,5minissuffcient.Bothhigherconcentrationandlong-termexposure

aredetrimentaltocellintegrityandsurfacemarkers.

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BoX2|PERFoRmSELECTIONDIRECTLYINCULTUREDISH•tIMInG1H40mlN

1.Culturecellsintomonolayerswithatleast90%confuence.

criticalstepPerformingselectiondirectlyintheculturedishmaintainscellsin

theirnaturalenvironmentandproteinsintheir

nativeconformation.Thereislessconcernofcellsurfacevariability.

2.Forthefrstroundofselection,usea100-mmx20-mmculturedish(cell

numberover5million).Washcellswithwashing

buffertwice.

criticalstepDonotuseaculturefaskwhenperformingselectiondirectlywith

adherentmonolayerbecauseitiseasiertowash

andremovethecellsfromanuncovereddishthanfromafask.

3.PrepareDNAlibraryin500plofbindingbuffer.

4.IncubatecellswithDNAlibrary.Useatotalvolumeof1,000plforthe100-

mmx20-mmculturedishandshakecontinuously

(50r.p.m.)oronslowrockerfor1h.

criticalstepBecausetheDNAlibraryvolumeisexpectedtocovertheentire

culturedish,adjustthetotalvolumeoftheDNA

libraryto1,000plwhilemaintainingthefnalconcentration.

5.Afterincubation,washcellsthreetimeswithwashingbuffer.

criticalstepEnsurethatthecellsarenotdetachedduringwashing,asthiswill

causelossofbindingsequences.

6.Add500plofDNAse-freewatertothewashedcells.Detachcellsusingcell

scraperandtransferthemintoa1.5-mltube.

7.Heatsuspensionat95℃for10minandrecoverthesuspendedDNAby

centrifugationat13,100gfor5min.Ensurethatthe

recoveryeffciencyisashighaspossible.

8.FollowSteps10-34toobtainthefrstselectedDNApool.

pausepointOnemaystoretheDNApoolat?20℃.

secondroundofselection

9.Usethegeneratedfrst-roundselectedpool,resuspendedinbindingbuffer.

10.FollowBox2,steps1-5.

11.Elutethecell-DNAcomplexin600plofbindingbuffer.Detachcellsusing

cellscraperandtransferthemintoa1.5-mltube.

HeatandrecovertheelutedDNAsuspensionbycentrifugation.

negativeselection

12.Usea100-mmx20-mmculturedishwithcellsinconfuence.

criticalstepAvoidnonconfuentcellcultureasDNAcansticktothebareculture

dish.

13.Washthecells.

14.Incubatecellswiththeelutedpool.

15.Afterincubation,recoverthebuffercontainingtheremainingsequences

thatdonotbindtothecontrolcells.Labelthisasthe

secondselectedDNApool.

pausepointOnemaystoretheselectedDNApoolat?20℃.

perprocedures—ssDna

16.FollowSteps15-34toobtainssDNA

subsequentroundsofselection

17.Usea60-mmx15-mmculturedishtocarryouttheincubationstepusing

250plofthessDNApoolwith250plofbindingbuffer

andmaintaininga1,000nMfnalconcentrationoftheselectedDNAlibrary.

Followwiththewashingandelutionsteps.

pausepointOnemaystoretheselectedDNApoolat?20℃.

Monitorprogressofselection

18.Onemaydissociatecellsandusesimilarbindingassaysasdescribedfor

suspensioncells(Steps52-59)oruseconfocalmicroscopy

whenadherent.

1174|VOL.5NO.6|2010|natureprotocols

thepurposeofselection.Ingeneral,anytemperaturebetween4and37℃is

applicableforthisprotocol;however,

highertemperaturessuchas37℃cancauseinternalization.Onceaptameris

selectedat4℃,itisimportanttotest

thebindingat37℃.Thisdoesnotmeanthatallaptamersselectedat4℃will

notbindat37℃.Fromobservation,most

oftheaptamerswillbindverywellat37℃,especiallythosewithveryhigh

affnity.Someoftheaptamersgeneratedat

4℃havebeenusedinvariousapplicationsat37℃(refs.30,33).Themost

importantpointhereistogenerateaptamers

withhighaffnitybygraduallyincreasingthestringencyofselectiontogenerate

aptamersthatcanbindatdiversebinding

conditions.Forinstance,aptamersthathavebeendevelopedusingDPBSas

thebindingbuffercanstillrecognizethetarget

inculturemedium.

criticalstepBecauseofthelargenumberofcellsrelativetothesmallvolume

of

incubationmedium,itissometimes

commontoseecellssettlingatthebottomofthetube,evenwhileshaking,so

occasionallycheckandresuspendthecells.

Washingstep•tIMInG40min

7|Afterincubation,centrifugecellsat150gfor3minat4℃.Remove

supernatantcontainingunboundsequencesand

resuspendcellpelletsin3mlofwashingbuffer.Shakeforabout30sand

centrifugeagainusingthesamecondition.Remove

supernatantcarefullywithtransferpipette,avoidingcellloss.Quicklyspin

down(30s)theresidualbufferonthewallofthe

tube.Toavoidcellloss,removeasmallamountofresidualbufferwithfolded

kimwipe.Repeatthewashingproceduretwo

moretimesforatotalofthreewashings.

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elutionofboundsequences•tIMInG15min

8|Forthefrstroundofselection,elutetheboundsequencesinwater.Add500

plofDNase-freewatertothecellpellet.

Resuspendcellsandtransfercellsuspensionintoa1.5-mlmicrofugetube.

criticalstepOnlyinthefirstroundisDNAelutedinwater.Insubsequent

rounds,DNAiselutedinbindingbuffer.Asthe

entireelutedpoolisamplifiedbyPCR,useonlyDNase-freewaterforthe

elutionoftheboundDNAsequencesduringthefirst

roundofselection.Otherwise,iftheDNAiselutedinDPBS,thesaltswithhigh

concentrationwillaffecttheefficiencyofPCR.

9|Heatthecellmixtureat95℃for10min,centrifugeat13,100gfor5min

andcollectsupernatantcontainingelutedDNA.

criticalstepDonotperformnegativeselectionatthispoint.Minimizetheloss

ofelutedsequencesduringthefirst

selectionround,aseachsequenceistheoreticallyrepresentedonlyonce,and

whenanysequenceislost,itcanneverbe

recovered.

pausepointStoreelutedDNAat?20℃.

perprocedures:peramplificationoftheentirefirstselectedpool•tIMInG1h

10|Thisstepisperformedforpoolsgeneratedfromonlythefrstselection.Setup

a1,000-plPCRamplifeationreaction

volumeasshownbelow:adjustthevolumeofwatertocompensateforthe

totalvolumeiftheDNAselectedpool

volumediffers.

reagentsreactionmixture(pl)

10xPCRbuffer100.0

dNTPmixture(2.5mMeach)80.0

FITC-andbiotin-primermixture50.0(0.5pMfinal)

DNAselectedpool(template)500.0

DNase-freewater270.0

HSTaqpolymerase3.0

11|Mixthoroughlyandpipet100plinto10individualtubes(using96-well

thermalcycler)for100plperreaction,orpipet

200plintofveindividualtubes(60-wellthermalcycler)for200plperreaction.

criticalstepNotethatthisstepisonlyperformedtostepupthecopiesof

individualsequencesandisnotnecessar­

ilypreparative.Toomanycyclesmayproducenonspecificamplicons,andthis

willaffectthepurityoftheDNAlibrarypool.

Choose8-10cycles.

12|PerformPCRamplifcation.Forexample,inprimerset2givenabove,use

thefollowingamplifcationconditions:denaturation

at95℃for30s,annealingat56.3℃for30sandelongationat72℃for30s,

followedbyfnalextensionfor3minat72℃.

criticalstepAlwayskeepTaqpolymeraseat?20℃untilreadytouse.Thaw

andkeepallotherPCRreagentsonice.

steptemperature(°c)time(s)

Hotstart95150

Amplification(10cycles)

Denaturation9530

Annealing56.330

Extension7230

Finalextension72180

Hold4

13|AfterPCRamplifcation,poolallreactionmixturestogether(total1,000pl).

Thereisnoneedtoperformagarosegel

electrophoresistoassesstemplateamplifcation.Inmostcases,thefrst-round

PCRamplifcationproductisinsuffcientfor

detectionbyethidiumbromide.

criticalstepNotethatthePCR-amplifiedpoolcanpotentiallycontaminatePCR

reagentsandprimers;therefore,keep

themseparated.

141LabelthePCRproductasthefrstselectedpool.

pausepointStoretheDNApoolat?20℃.

perprocedures:determinetheoptimumnumberofcyclesforpreparativeper

•tIMInG1h30min

15|ThetotalPCRreactionmixturevolumeforeachtubeis50pl,withthe

amplifedfrstselectedpoolat10%servingas

thetemplate.ChoosePCRsamplesatthefollowingcycles:4,6,8,10and12,

andonenegativecontrolatthetwelfthcycle.

AddthefollowingreagentstoPCRtubesasshownbelow:

reagentspositivereactionmixture(pl)control(pl)

lOxPCRbuffer25.05.0

dNTPmixture(2.5mMeach)20.04.0

FITC-andbiotin-primermixture12.5(0.5pMfinal)2.5

AmplifiedDNAselectedpool(template)25.0(10%ofreactionmixture)—

Supernatantofpurecelllysate—5.0

DNase-freewater166.753335

HSTaqpolymerase0.750.15

Abbreviation:FITC,fuoresceinisothiocyanate.

16|Mixthoroughlyandpipet50plofthepositivereactionmixtureintofve

individualtubes.

17|PerformPCRamplifcationusingthePCRprogramdescribedinStep12with

themaximumnumberofcyclesas12.

18|Openthethermalcyclerandtakesamplesatthespecifedcyclesasshown

inStep15.

perprocedures:agarosegelelectrophoresis•tIMInG1h40min

19|Prepare3%agarosegel.

20|Prepareagarosegelsamplesasshownbelow:

positive(pl)control(pl)ladder(pl)

Blue/greenloading

dye(6x)

222

PCRproduct10-----

PCRnegativecontrol—10—

Ladder(25bp)-----1

H2

O——9

21|Loadsamplesinlanesandperformelectrophoresisat100Vfor40min.

22|Removethegel,stainwithethidiumbromide,andobservethebandsunder

UVlightandtakeimageofthegel(Fig.2).

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23|Selectacyclenumberthatyieldsabrightbandwithoutnonspecifc

amplicons.OnthebasisofFigure2,agoodexample

isthetenthcycle(lane5).InpreparativePCR,usethiscycleofamplifcationto

producemorePCRproductsfortheprepara­

tionofssDNArequiredforthesecondroundofselection.

pausepointOnemaystorethelibrarypoolat?20℃.

perprocedures:preparativeper•tIMInG2h

24|Usethesamereagentconcentrationsaslistedin

Step15,butinlargervolumes.Forthesecondroundof

selection,1,000ploftotalPCRreactionmixtureisenough

togeneratetherequiredamountofssDNA.Moreover,the

entiressDNApoolthatisgeneratedhereisusedforselec­

tion,whereasinthehigherrounds,someareusedformonitoringtheselection

progress.PreparePCRreactionmixtureas

shownbelow:

reagentspositivereactionmixture(pl)

10xPCRbuffer100.0

dNTPmixture(2.5mMeach)80.0

FITC-andbiotin-primermixture50.0(0.5pMfinal)

AmplifiedDNAselectedpool(template)100.0(10%ofreactionmixture)

DNase-freewater670.0

HSTaqpolymerase3.0

Abbreviation:FITC,fuoresceinisothiocyanate.

1234567Figure2|Agarosegelelectrophoresisimageshowingtheproducts

ofthevariouscyclesofselectedDNAlibraryamplification.Lane1=25-bpladder;

lane2=4cycles;lane3=6cycles;lane4=8cycles;lane5=10cycles;

lane6=12cycles;andlane7=negativecontrol.

criticalstepItisnotnecessarytoperformcontrolPCRforthepreparative

processoncethecycleoptimizationcontrol

dataareclean.

25|Mixthoroughlyandpipet100plor200plofthemixtureintotenPCRtubes

(96-wellthermalcycler)orfvePCRtubes

(60-wellthermalcycler).ClosethecapsecurelyandperformPCRusingthe

programdescribedinStep12,butwiththe

chosennumberofcycles.

26|AfterPCR,poolallPCRproductstogetherandperformgelelectrophoresis

(Step20)toconfrmthePCRproduct(Fig.3).

pausepointOnemaystorethelibrarypoolat?20℃.

preparationofssDnafromperproduct•tIMInG40min

27|InsertaflterandsecureitfrmlyatoneendofanemptyDNAsynthesis

column.Removetheplungerfroma10-ml

syringewithoutaneedleandinserttheemptysyringeattheotherendofthe

DNAsynthesiscolumn.

28|Mountthesetuponaclamp.Add200plofstreptavidinSepharosebeads

suspension.Inserttheplungerandallowthe

storagebuffertodrain.

29|Washthebeadswith2.5mloflxDPBS.

30|PassthePCRproductthreetimesthroughthe

column.Washthebeadsagainwith2.5mlofDPBS.

31|Add500plof200mMNaOH,inserttheplunger

andcollecttheeluatecontainingtheFITC-labeled

ssDNA.

criticalstepAllowthePCRproductandtheNaOH

solutiontopassthroughthecolumngradually.Pushingthe

plungerhardwillreducetheeffciencyofthessDNAyield.

Furthermore,donotremovetheplungerfromthesyringe

21

Figure3|AgaroseelectrophoresisgelimageofpreparativeselectedDNA

librarypool,confirmingtheefficiencyofamplification.Lane1=25-bpladder

andlane2=PCRproductof10cycles.

whilethesyringeisconnectedtothecolumncontainingthebeads.Always

disconnectthesyringefromthecolumnbefore

removingtheplunger.

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DesaltingssDna•tIMInG30min

32|WashtheNAP5columnwithatleast15mlofdeionizedwater.Add500pl

ofelutedssDNA.Allowtheentiresample

volumetodrainandthenelutethessDNAwith1,000plofDNase-freewater.

criticalstepAlwayskeepthesurfaceofthedesaltingcolumnmoist.

trouBlesHootlnG

QuantifyingelutedssDna•tIMInG5min

33|DeterminetheconcentrationofssDNAbyUVabsorbanceat260nm.

DryingssDna•tIMInG3h

34|ConcentratethessDNAusingaDNASpeedvacdryer.Technically,thisstep

istheendofoneroundofDNAaptamerselection.

pausepointOnemaystorethedriedssDNAat?20℃andresuspenditin

bindingbufferwhenneededforthenextround

ofselection.

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secondroundofselection•tIMInG2h30min

35|ResuspendssDNAinbindingbuffertoobtainaconcentrationof1,000nM.

36|DenatureandrenaturethessDNApoolasdescribedinStep2.

preparationofcellsforsecondroundofselection

37|FollowthesameprocedureusedinSteps3and4butwithlessthan5

millioncells.Washing

38|RepeatStep5.

Dnaincubation

39|Add200plofssDNA(Step36)to200plofcellsuspension.RepeatSteps7,

8(use600plbindingbuffer)and9.

pausepointOnemaystoretheselectedDNApoolat?20℃.

negativeselectionprocedure

cellviabilityandnumberofsuspensioncells•tIMInG2h30min

40|CheckcellviabilityandcountcellsasdescribedinSteps3and4.Useat

least10millioncontrolcells.

criticalstepTheviabilityofcontrolcellsisimportantinnegativeselection

becausesequencesthatnonspecifcallyadhere

toandenterdeadcellswillcausethelossofspecifcsequences.Totheextent

possible,usemorethan95%viablecells.

41|WashcellsasdescribedinStep5.

42|IncubatecellpelletwiththeentireelutedDNApoolfrompositiveselection

onicewithcontinuousshakingfor1h.

43|Afterincubation,spindownthecellscontainingthebindingsequencesat

3,059gfor5min.

441Recoverthesupernatantcontainingsequencesthatdonotbindtocontrol

cellsandlabelthispoolasthesecond

selectedDNApool.

pausepointOnemaystoretheselectedDNApoolat?20℃.

peramplifeations

Determinationoftheoptimumnumberofcyclesforpreparativeperofthe

secondselectedDnapool•tIMInG3h10min

45|Estimatetheminimumandmaximumnumberofcyclestobeassessedon

thebasisofthepreviousPCRcycle

optimizationprocedures(Steps15-23),butusethesecondselectedDNApool

asthetemplate.Reducethenumberofcycles

tobeassessedtoaboutfourandmaintaincycleintervalsoftwo.

protocol

preparativeper•tIMInG2h

46|FollowthesameprocedureasdescribedforSteps24-26

(Fig.4,panelb).

pausepointOnemaydrytheselectedDNApoolandstore

itat?20℃.

ssDnapreparation•tIMInG40min

47|FollowSteps27-31.

Desalting,quantifyinganddrying•tIMInG3h35min

48|FollowsSteps32-34.

subsequentroundsofDnaaptamerselection•