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1、1ProtocolfGrowthofHEK293CellsinSuspensionAlwayswearlabcoatglovesBuffersMedia(seealsofilecalledmedia_buffers.doc)Detergents:Dodecyl?1maltoside(DM)noctyl?Dglucoside(OG)canbeobtainedfromAnatrace(MaumeeOH).ProteinASepharosew

2、asfromRepliGen(CambridgeMA)CNBractivatedSepharose4BwasfromPharmacia.DMEM(DulbeccosmodifiedEaglesmedium):StardDMEM:StardDMEMmediumHBGrowaspurchasedfromIrvineScientific(SantaAnaCA).Powderf10Lwillbedissolvedin9LMillipewater

3、(distilledwater).Addsodiumbicarbonateifrequired(checkmanufacturer’smanualpowdercontainer).Stiruntildissolvedbutavoidairbubbles.Fillupto10Lsterilefiltrateinto20autoclaved0.5Lbottles.Befefirstuseofabottleadd10%(vv)heatinac

4、tivated(30min56C)FetalBovineSerum(FBS)penicillin(100unitsml)streptomycin(100?gml).Youmayhavetoaddglutamineaswell(checkmanufacturer’smanualpowdercontainer).Steat4C.CustommadeDMEM:CustommadeDMEMlackingtheaminoacidsofchoice

5、sodiumpyruvatecalciumsodiumbicarbonatewasobtainedfromAtlantaBiologicals(NcrossGA).Steat4C.DMEMwasmadeoutofitscomponents(e.g.Sigmacellculturetested).Steat4C.TherearethreetypesofDMEMusedinthisprocedure.TheDMEMwith1.4mMCais

6、theDMEMusedfthestepsupuntilthesplittinginto20plates.ThesubsequentstepsuseDMEMwith680uMCa340uMCa.DMEMisusuallyprepared23Latatimewith1.4mM340uMCastedinsterilebottlesat4C.The680uMcanbeobtainedbymixingthetwoDMEMmedias.Antibi

7、oticsproteaseinhibits:Geicin(G418)trypsinEDTApenicillinstreptomycinwereobtainedfromGibcoBRL(GaithersburgMD).AprotininbenzaineleupeptinpepstatinAwerepurchasedfromBoehringerMannheim(IndianapolisIN).Serum:Fetalbovineserum(F

8、BS)cellculturegradecalciumchlidePluronicF68PMSFaminoacidswerefromSigma(St.LouisMO).FBS(500mL)washeatinactivated(56?C30min)dialyzed(1kDacutoff)threetimesagainst10LPBSbuffer(137mMNaCl2.7mMKCl1.5mMKH2PO48mMNa2HPO4pH7.2.)ove

9、r3daysat4?C.3withdifferentamountsofgeicin)fromstocksolution.TherestofthesolutionisDMEM.(Fexample0.4mLG418stocksolution9.6mLDMEMsolution).Pipettethesolution(DMEMcells)updownintheconicaltubetoresuspendbreakupthevisiblepell

10、et.Pipettethesolutionontoa10cmculturedish.Addthecalculatedamountofgeicinontotheculturedish(e.g.0.4mL)swirltomixthesolutions.GeicinfcesthecellstoretaintheextraDNA.Placethesolutiononaninvertedmicroscopecheckfsinglecells.Th

11、esecellsshouldmovewhenthedishisswirled.i.e.theyshouldnotbefloatingclumpsofcellsattachedtothebottomoftheplateatthistimepoint.Placethecoveredculturedishina37Cincubatat5%CO2.(Incubatneedstobecalibratedatthestarttomakesureit

12、equilibratesat5%CO2.)Checkifthecellssinktothebottomofthedishinonetwohours.Cellsthathavesunktothebottomshouldnotmovewhenthedishisswirled.Itisnmalthatsomecellsmaynothaveattachedyet.DisposeusedtubespipettesinbiowastereturnD

13、MEMto4Cgeicinto20C80Cfreezers.Cellsshouldbegrownfapproximatelyfourdaysfedonceduringthedleofthistimeperiod.FeedingthecellsinvolvesremovingtheoldmediumaddingnewDMEMgeicin(ionmarker).Prepareallnecessarymaterialsaheadoftimei

14、nthehood:pipettesgeicinDMEM.DMEMshouldbewarmedto37Cinawaterbathgeicinthawedina4Crefrigeratatroomtemperature.Removetheculturefromtheincubat.Tiltthedishvacuumthemedia(usesterilepipettes)fromthesideofthedish.Thecellsshoulda

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