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CHO Centrosome Prep
蚂蚁淘
/发布时间:1631208604 /浏览量:329
ArshadDesai
4/94
Cells:
WegrowourCHOswithMEM[[alpha]](withoutnucleosides)+10%BovineCalfSerumandpenn/strep/glutamine.Foraprepitisbesttogrowtwentylargeplates(150mM)andthecellsshouldbegrowntooverconfluence-tilltheystartpilinguponeachother(youwillneedtofeedthemfrequentlytokeepthemhappy)
ProtocolRationale:
TheprotocolisidenticaltoTim"spublishedinVol134ofMethodsinEnzymology.ThekeystepisthelysiswhichsolubilizescentrosomesawayfromnucleibyverylowionicstrengthlysisaftertreatmentofcellswithnocodazoleandcytochalasinB.ThereleasedcentrosomesarethencentrifugedontoaFicollcushion(toavoidpelleting)andtheinterfacebetweenthelysateandtheFicolliscollectedandthecentrosomesareconcentratedonasucrosegrADIent.FractionsareassayedbyspindownanddoubleIFwith5051serumandanti-tubulinandthepooledfractionsarefrozeninliqN2.
Whatyouneed:
Equipment:
- refractometer
- SorvallwithcoldHB-4
- UltrawithcoldSW27
- Somesortofgradientfractionatorwithafractioncollector
- 1630mlcorextubes(cold)
Solutions:
Alotofthesesolutionsarew/w;Timsaysthattomaketheseweighoutsucroseandthenaddbuffertillweightis100g.
Wash&Lysis:
- PE:10mMPIPES,1mMEDTA,8mMBME
- Makea50XstockandpHto7.2withKOH
- LB:1mMTris-HCl,8mMBME
- MakeTrisas2MstockandpHto8.0withHCl
- LB+0.5%NP-40:
- (warmfor30"to37deg.CtoensureNP-40hasdissolved)
- PBS:130mMNaCl,2mMKCl.8mMNa2HPO4,2mMKH2PO4
- Makea10Xstock
Nightbefore:
Make600mlof
- 1XPBS
- 0.1XPBS
- 0.1XPBS,8%(w/w)ultrapuresucrose
- 8%(w/w)ultrapuresucrose
- LB(w/oBME):add280ulBMEbeforeuse
- LB+0.5%NP-40(w/oBME):add280ulBMEbeforeuse
andputallthesebuffersincoldroom
SucroseGradients:
Useultrapuresucrose
- 20%(w/w)and62.5%(w/w)sucrosein1xPE+0.1%TX-100.Make100gramsofeachasfollows:
- Weighoutsucroseandthenonthebalanceadd1XPE+0.1%TX-100tillweightis100grams.
Justbeforepouringgradientsadd28ulBME/50g
Pourgradientsnightbeforeorduringdrugtreatment(seelater).Ifinditeasiesttopournightbeforeandstoreincold.
- Topourgradients-firstputa5mlheavysucrosepadandthenpourgradientontopofthat.FortheprepoutlinedbelowIpour2gradientsinSW27tubes:
- 4mlheavysucrosepad(use62.5%orhigher).
- 16mlgradient.
Thecentrosomesareveryclosetothebottomofthisgradient.Thepadeliminatesthemfromenteringthecurveofthetubeandalsogivesalittleleewayinsettingupthefractionation.
Ficollcushion:
20%(w/w)Ficoll(MW400,000)in1XPE+0.1%NP-40
- MakeupPE+0.1%NP-40(noBME).
- Weighout10gFicoll.
- AddPE+0.1%NP-40tilltotalweight=50grams.
- StiratRTforseveralhourstodissolveandstorecold.
- Beforeuse,add28ulBME
Protocol:
- Warmup300mlofCHOmediumto37deg.CandaddcytochalasinB(150ulof10mg/ml)andnocodazole(300ulof10mg/ml)
- Makesureallbuffersareincoldroom,thereisrockerinthecoldroomandthereisagoodaspiratorinthecoldroom.HookupasawedoffpipettotheaspiratorandmakesurethereisaLARGEtrap(atleast4L)
- Addmediumwithdrugsto10platesofcells.Addmediumwithdrugstotheother10plates45"later.
- After90"indrugmediumprocessfirst10plates:bringtocoldroomandwashwiththefollowingbuffers:
- 1XPBS
- 0.1XPBS,8%(w/w)sucrose
- 8%(w/w)sucrose
- LB
- andthenpipeton
- LB+0.5%NP-40(10mls/plate)
(Thesewashesmustbedonerapidly-allwashesshouldbeunder1"perplate.ThewayIdothemistopouronthewashbufferfromabeakerorgradcylinder(~30mls),immediatelyrockbackandforthandaspirateASAPbeforepouringonthenextwash.Itiscriticaltodothisquicklytogetgoodlysisoryouwilllosemostofthecentrosomesinthenuclearpellet).AfteraddingtheLB+0.5%NP-40transfertheplateontoarockerinthecoldroom.
- After10"pipetoffthelysateintoa30mlcorextube.Add1/50volof50XPE(0.5mlfor25mls;withBME)
- SpintubesinHB-4for3"at3000rpmat4deg.C
- Transfersupetoafreshcorextubeandunderlaywith2mlofFicollcushion(Iusea6ccsyringewith16gaugeneedlewithathinpieceoftygontubingwhichIcanslidetothebottomofthetube).
- Spinat12,700rpmfor15at4deg.Cfor15".Assoonasspinisstarted,processthenextsetofplatesthroughsteps4-7.
- Aspiratesupetillapprox.2mlabovecushionandthencollectinterfacewithapasteur-seeTim"sprotocol.Collect~2ml/tubeandpool.CheckFicollconcentrationbyrefractometryanddiluteto10%(w/w)orlower-necessarytomakesureitlayersontothesucrosegradientanddoesn"tsink.
- Finishcollectinginterfacesfromsecondsetofplatesandthenpoolallcollectedinterfaces,ensureFicollis<10%(w/w)andloadonto1gradient.Spin1hr-1hr30",SW27at2deg.C.
- Fractionategradientfrombottom-0.3-0.5mlfractionsandreadsucroseconcentrationbyrefractometry.Assayfractionsbetween48and60%(w/w)sucrose.5uloffraction+5mlPE-mixwellandpelletontocoverslips:12,500rpmfor15"at4deg.CinHB-4.Postfixinmethanol(-20deg.C)for5"andrehydrateanddo5051+antitubulinfollowedbyanti-mouse,anti-humansecondaries.Assesspeakbyconcentrationofdoublestainingdots,poolandfreezeinliqN2in10ulaliquots.
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