细胞结合实验.docx

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1、细胞结合实验细胞结合实验1Preparece11sfortheexperiment.Thisprocedurewi11presentthedetai1edce11-SE1EXprotoco1forbothsuspensionandadherentcu1turedce111ines.Foradherentce11s,therearetwooptions:theexperimentcanbeperformedeitherdirect1yinacu1turedishorusingdissociatedce11s.Ifusingdissociatedce11s,preparethembyfo11owi

2、ngthestepsgiveninBox1beforefo11owingtheproceduregivenbe1ow.Ifperformingtheexperimentinacu1turedish,refertoBox2andcontinuewiththeprocedurefromStep61.Initia1Dna1ibrarypoo1preparationtIMInG20min准备实验细胞。这个程序将详细的细胞SE1EX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

3、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2Add201of0.5mM(10nmo1)DNA1ibraryto3501ofbindingbuffer,mixandheatthemixtureat95for5min.Snap-coo1oniceandkeeponiceunti1readytouse(sameday).pausepointIfse1ectionisnotaccomp1ishedonthesameday,storetheDNA1ibrarypoo1at?20.Thaw1ibraryonicewheneverreadytouse.Thedenaturations

4、tepisnotnecessaryonceDNAhasbeenthawedonice.critica1stepHeatingtheDNAat95andsubsequentfastcoo1ingoniceareimportanttocreatefo1dedssDNA.preparationoftargetce11s:ce11viabi1itytIMInG30min添加2010.5毫米(10nmo1)3501的结合缓冲液DNA文库，混合和热的混合物在95P5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?2(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DN

5、A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3Determinece11viabi1ityusingTrypanb1ueexc1usionassay.critica1stepCe11viabi1ityassessmentisveryimportant,especia11yforsuspensionce11s.Toomanydeadce11swi11serious1yaffecttheefficiencyofse1ection.DNAcannonspecifica11yadheretoandenterdeadce11

6、s.Thiswi11causethe1ossofimportantsequences,de1ayofenrichmentoreventhefai1ureofse1ection.Asthehighestce11viabi1ityisidea1,itispreferab1etousemorethan95%viab1ece11s.preparationoftargetce11s:ce11numberHMInG20min4Determinetheconcentrationofce11susingahemocytometer.Onthebasisofce11count,determinewhatvo1u

7、mewi11correspondtothenumberofce11sneededforaspecifcroundofse1ection.critica1stepForthefirstroundofse1ection,usethehighestnumberofce11spossib1eforco11ectingspecificsequences,preferab1ybetween5and10mi11ionce11s.preparationoftargetce11s:washingbycentrifugationUMInG30min5Takethece11vo1umethatyie1dsthede

8、sirednumberofce11sintoa15-m1centrifugetubeandcentrifugece11sat150gfor3minat4.Removethesupernatantandadd3m1ofwashingbuffer.Resuspendce11sbypipettingupanddownztappingthebottomofthecentrifugetubeormi1dvortexing.Pe11etthece11satthesamespeedandduration.Repeatwashingoncemore.critica1stepAvoidstrongvortexi

9、ngasitcancausece11breakageandmayeventua11yaffectse1ection.Incubationofce11swithDna1ibrarypoo1tIMInG1h6Resuspendce11sin3301ofbindingbuffer.Adda11ofthe3701snap-coo1edDNA1ibrary(Step2)tothe3301ce11suspension,mixthorough1yandincubatethemixtureonicefor1honarotaryshaker.IncubationtemperaturedependsonBoX1I

10、DISSOCIATEADHERENTCE11SHMInG4H15mIN1. Sp1itce11s24hbeforedissociationtreatment.2. Dissociatece11sfromcu1turefaskordishwithnonenzymaticdissociationso1utionorbyshort-term(30s-1min)trypsintreatment.Afterincubation,removetrypsinordissociationbufferandimmediate1yaddco1dcu1turemediumcontainingFBS.Ce11sare

11、sp1it24hbeforedissociation;hence,theycanberemovedbyshort-termtreatment.Ifce11sare1eftfor2-3d,thiswi11notbepossib1e.3. Transferce11sfromfaskordishtocentrifugetubeforsubsequentuse.FBSwi11ha1tfurtheractionofresidua1trypsin.4. Force11sdissociatedwithnonenzymaticdissociationbuffer,countce11sanduserequire

12、damountforaptamerse1ectionbyfo11owingthesameprocedureasdescribedforsuspensionce11s(Steps2-60).5. Force11sdissociatedbyshort-termtrypsintreatment,usece11simmediate1y,asdescribed(Steps2-60),orincubatece11sunderce11cu1tureconditions,butwithrockingforat1east2h.critica1stepContinuousrockingwi11preventthe

13、ce11sfromstickingtothebottomofthefaskordish.Itisimportanttocu1turece11sforat1east2htorecoversomeoftheproteinsthatmighthavebeenaffectedbytheshort-termtrypsintreatment.6. Afterincubation,countthenumberofce11sandusethemforse1ection,fo11owingthesameprocedureasdescribedforsuspensionce11s(Steps2-60).5. Fo

14、rce11sdissociatedbyshort-termtrypsintreatment,usece11simmediate1y,asdescribed(Steps2-60),orincubatece11sunderce11cu1tureconditions,butwithrockingforat1east2h.critica1stepContinuousrockingwi11preventthece11sfromstickingtothebottomofthefaskordish.Itisimportanttocu1turece11sforat1east2htorecoversomeoft

15、heproteinsthatmighthavebeenaffectedbytheshort-termtrypsintreatment.6. Afterincubation,countthenumberofce11sandusethemforse1ection,fo11owingthesameprocedureasdescribedforsuspensionce11s(Steps2-60).critica1stepTrypsintreatmentmustbeperformedwithextremecautionbecausesubstantia1damagetosurfaceproteinsca

16、noccurifthetreatmentispro1onged.Moreover;donottreatce11swithpro1ongedexposuretotheharshnonenzymaticdissociationbuffer.Atthemost,5minissuffcient.Bothhigherconcentrationand1ong-termexposurearedetrimenta1toce11integrityandsurfacemarkers.trouB1esHoot1nGBoX2IPERFoRmSE1ECTIONDIRECT1YINCU1TUREDISHHMInGIH40mIN1. Cu1turece11sintomono1