ProcedureI.INTRODUCTIONThe293EBNAcelllineisestablishedfromprimaryembryonalhumankidneycellstransformedwithshearedhumanadenovirustype5DNAandEpstein-Barrvirusnuclearantigen1,allowingepisomalamplificationofplasmidscontainingtheviralEBVoriginofreplication.Thisprotocoldescribestheinstructionsforthegrowth,maintenanceandtransfectionofsUSPensionadapted293-EBNAcelllineusingthePEIexpressionsystem.CellscanbepurchasedfromATCCII.REFERENCES•Transfectol-LSMammalianTransienttransfectionKit.GeneChoice;TR10v2KT-011106III.EQUIPMENT,MATERIALS,ANDREAGENTS•CO2incubator,FormaScientificincubator•BioSafetyCABInet,•OrbitalShaker,FormaOrbitalShaker•WaterBath,VWRScientific•500mlVentedCapErlenmymerFlask,•125mlVentedCapErlenmymerFlask,Corningproduct•Pro293S-CDM,1L(CambrexBiosciences,Cat12-765Q)•Penicillin-Streptomycin,Invitrogen•TransfectionReagent,TR-1010-LiterTransienttransfection•BrightLineHemacytometer,Sigma,•TrypanBlueSolution(0.4%),Sigma,•0.22umGPExpressMembraneFilter,MilliporeIV.GENERALCONSIDERATIONSA.PriortoStartupSince293cellstendtoaggregateinsuspensionculture,293-EcellsareculturedinCambrexPro293suspensionchemicallydefinedmediasystem(Pro293s-CDM),whichhelpssupportsinglecellsuspensions.MediadoesnotcontainL-Glutamineandshouldbeaddedpriortouse.Whiletheuseofantibioticsinantibodyproductionisnotrecommended,theiruseshouldbeconsidereddependentondownstreamapplication.Antibioticsshouldbeusedinculturemaintenance(5ml/LPenicillin-Streptomycin).Forgeneralcellmaintenancesplitcellswhentheyhavereachedadensityof1-3x106cells/ml(every3to4days).TrypanBlueexclusionisusedtodetermineviabilityanddensity..V.PROCEDURESA.ThawingandEstablishingCells1.Prepare1LPro293Smedia(5mlsPenicillin-Streptomycinand10ml/LL-Glutamine)2.RemovecryovialfromLiquidNitrogenandquicklythawin37oCwaterbath.3.Add19mlspre-warmedcompletemediato125mlpolycarbonateshakerflask.4.Whencryovialhasthawed,decontaminateoutsideofvialwith70%ethanol.5.Transfercellstoshakerflaskcontainingcompletedmediaunderbiosafetycabinet.6.Incubatecellsinincubator(37oC,8%CO2)onorbitalshakerrotatingat125RPM.B.PassagingCellsSubculturecellswhendensityisapproximately1-3X106viablecells/ml,whichtypicallyoccursevery3to4days.Inorderforcellstoproperlyrecoverafterthawingcellsshouldbesubculturedfortwopassagespriortouseintransfection.1.DeterminecelldensityandviabilityusingTrypanBlueexclusionMethod(seeMethodbelow)2.Usingcelldensitydeterminedinstep1,calculatethesplitrationeededtoseedthenewshakerflaskat3x105cells/mlaccordingtothedesiredfinalvolumeofshakerflask(3x105cells/ml)(desiredfinalvolume)/celldensity=cellvolumeneeded3.Transferappropriatevolumeofcellstocorrectfinalvolumefreshmedia(desiredfinalvolume)–(cellvolumeneeded)=freshmediavolume4.GentlyPipettecellstobreakupcellclusters5.Returncellstoincubator(37oC,humidified8%CO2,orbitalshaker125RPM).6.RepeatstepsasnecessarytomaintainorexpandcellsC.FreezingCellsCellsmaybefrozendirectlyinPro293Smedia+10%DMSO.Freshmediashouldbeusedwhenfreezingcellsandcelldensityshouldbe5-8x106cells/ml.1.Continuetogrow293Ecellsuntiltheirdensityreaches1X106cells/ml.2.Determinecelldensityandviablecellcount(page4)andcalculatethevolumeofcellsneededtoyieldafinaldensityof5X106cells/ml.3.Transfercellstoconicaltubesandcentrifuge100xgfor5minutes4.Prepareenoughfreezingmedia(90%Pro293Smedia+10%DMSO)toresuspendcellsatafinaldensityof5X106cells/ml.5.Aliquot1mlofcellsuspensiontocryovialsonice.6.Transfercellsto-70oCovernight7.Transfercellstoliquidnitrogenfreezerforlongtermstorage.D.TransfectingCells293Ecellscanbetransfectedwithavarietyofmethods.DescribedbelowistransfectionusingTransfectolLS(GeneChoice).Transfectionexperimentscanbemodifiedbyscalingvolumesupordownasneeded.Thefollowingconditionsdescribedareusedfor200mltransfections.Changingculturemediaisnotrequiredaftertransfectionandallreagentsshouldberoomtemperaturepriortouse.Transfection:1.DeterminecelldensityandviabilityusingTrypanBlueexclusionMethod(seepage4)2.Ifyouhaveenoughcellscollect293Ecultureinanappropriatevesselandcentrifugeat1000RPMfor5minutes3.Aspiratemedia(Becarefulnottodisturbpellet)andresuspendcellsinfreshPro293Smedia(bygentlepippeting)tocelldensityof1x106cells/ml.a.Foroptimalresultsrecountcells,viability(shouldbegreaterthan90%),andmakesureyouhavesinglecellsuspension.Returnculturetoincubator.4.30minutespriortotranfectionadd5mLEnhancerLS(BottleA)5.Dilute200µgDNAintotal10mlvolumeDiluentLS(BottleB),mixgently6.Add1mlTransfectolLS(BottleD),mixgently7.IncubateRoomTempfor15-20minutes8.AddtotalvolumeofDNATransfectol-LSComplextocellcultureflask.Swirlflasks.9.Replacecellcultureto125RPM,37oC,8%CO2incubator.10.Collectafter6-8days.a.YoumaywanttoaddsomefreshmediaataboutthehalfwaypointMediaCollection:1.Collectcellculturemediabycentrifugationat5000RPM,for15minutes.2.Sterilefiltermediaover0.22?mGPExpressMembranefilter3.Storemedia4oCpriortoantibodypurificationVI.ASSOCIATEDPROCEDURESA.Trypanblueviabilitytest1.Transfer0.9mlof0.4%TrypanBlue+0.1mlofcellsuspensioninanEppendorfTube2.Gentlymixtoavoidcellclustersbybypipettingupanddown3.Allowtostandfor2minutes(viablecellswilltakeupdyeifincubationgoestoolong)4.Pipetenoughofmixtureintocoverslippedchambersofhemacytometertofill5.Countviableandnon-viablecellsin5squares.Foroptimalresults,adjustcelldensitytobetween20-50cellspersquareCalculations:Viablecells/ml:thenumberofviablecellsperquadrant×dilutionfactor×104cells/ml(e.g.1:10dilution,200cellscountedin5squares=200/5=40cellsperquadrant×10×104cells/ml=4×106cells/ml).cells/ml×originalvolume=Totalcells(e.g.40millioncellsin10ml)cellviability(%):totalviablecells(unstained)/totalcells(stainedandunstained)×100(e.g.20stainedcellsperquadrant:50%viability)

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