HealthySp2/0cellsshouldberapidlygrowingbythistime.Sp2/0cellsshouldbestartedabouttwoweeksbeforethecellfusion.Everytwodays,theyshouldbecentrifugedata64.4xgontheIECclinicalcentrifuge(in50mltubes)fortwominutes.ThemediashouldberemovedandthecellsresUSPendedinfreshmedia.Thisensuresthatonlythehealthiestcellswillfalltothebottomofthetube.Centrifugationathigherspeedsorforlongertimeperiodsshouldbeavoidedatthisstep,becausethatcausesweakoroldSp2/0cellstoprecipitatealso,andiscontra-indicativeofwhatyouwanttoaccomplish.Beginthecellfusionbycentrifugingtwo50mloreight10mlflasksofSp2/0cellsthathavebeentransferredtofreshmediaonetotwodayspreviously,atasettingofnotmorethan3fornotmorethan3mininanIECclinicalcentrifuge.Decantthemedia.Youneedaratioof1:5healthy,rapidlygrowingSp2/0tospleencells.Thisnumberofflaskswillprovideaboutthecorrectnumber.Removetwospleensfromthemiceselectedabove,andplacetheminwashingmedia.Teaseapartthespleens.Dontbeafraidtotaketwopairoftweezers,ortweezersandsyringetipandshredthespleens.Thenremovecellsbyperfusingmediathroughthespleensandalsobydisruptingportionsofthespleenwiththetweezers.Thenremovetheremaininglargechunksofspleenswithtweezers,andplacethecellsina50mltube.Centrifugeatasettingof3inIECclinicalcentrifugefor3minutes.Usingwarm(37oC)washingmedia,transferallthecellstoone50mltube,bringthefinalvolumetoabout25to30mls,mixbyvortexing(THISISTHELASTTIMEYOUWILLVORTEXANYTHINGinthisprocedure),andcentrifugeatasettingof5for3minutes.Theobjectivehereistopackthecellstightlysothattheircellmembranesaretouching.Youalsoneedalargesurfacearea,andtopreventlayeringofthelargeSp2/0cellsonthebottomandspleencellsontop.Thebesttubestousearethewidest,flattestbottomedones(our50mlconicaltubesworkfine).Theworsttubesarethenarrow,veryconicalbottomedtubes(our15mlculturetubes).Ifitisnecessarytouse15mltubes,useroundbottomonesonly!Addonemlofwarm(37o)50PEG(m.wt.1500)(sourceandtype)overoneminute,gentlyswirlingthetubeat37oC.IfyouaddthePEGwitha1mlPipetteandgentlystirthecells,youwillprobablyobtainmanymorehybridomas.AfterthePEGisadded,capthetube,gentlyswirlacouplemoretimes,thencentrifugeatasettingof5for3minutes(minimum).Thisisthecriticalstep.Cellsmustbecompactedandstucktogetherforcellfusiontooccur.ThisstepwillalsoensurethatthePEGcomestothesurfacewhilethecellsarecompactedatthebottomofthetube.ThenremoveasmuchPEGaspossIBLewithapipette.(CAUTION:PEGistoxictocells,andshouldberemovedpromptly.Thisisnotthestepto\"takeabreak.\")Add8to10mlsofwarm(37oC)washingmediaoveraperiodof2.5to3minutes,againgentlyswirlingthetube.Donotdisturbthecellpellet.Addthemediaasgentledrops.Rememberthatthecellsareveryfragile.Afteradditionofthemedia,capthetube,gentlyswirlitanothertimeortwo,thencentrifugeatasettingof5for3minutes.Removethemedia.Thisstepcanberepeatedoncemore,toensurethatallthePEGisremoved,butthisappearstobeanoptionalstep.Finally,usingalargetippedpipette,suchasa5mldisposablepipette,andwarmcompletemedia(containingHTmedia,fortheverybestresults)transferthecellpellettoa50mlflask.Donotdisruptthecellpelletoruseasyringe!.Hybridomaswillgrowoutoftheclumpsofstuck-togethercells.Allowthecellstogrowina50mlflaskinthepresenceofCO2,HTmedia,andtheunfusedcells,for4to24hours.Itseemsthatthecellsaretoofragiletohandleimmediatelyafterfusion,butlargenumbersofhybridomashavebeenobtainedwhencellswereplatedonthesamedayasthecellfusion.ColdSpringHarbor(1989)doesnotincludetheovernightincubationpriortoplating.AddHATmediatothe50mlofmediaintheflask(50xHATisusedataratioof1ml/100mlofmedia;500xHATisusedataratioof.1ml/100mlofmedia).Platethe50mlsintothecenter60wellsof596wellplatesWITHAWIDETIPPEDPIPETTE(300wells)-DONOTuseasyringeandneedle!Cellsshouldcoverthebottomsofthewells.ThisisimportanttomaintaintheCO2levelatahighenoughlevel,andpossiblythehybridomasobtaingrowthhormonesfromtheunfusedspleencells.Thecompletemediasupplementedwithfetalbovineserumshouldsupportthegrowthof50cells/well,butoursdoesnotappeartodothis.Thefirstthingonewouldconsider,isthatthefetalbovineserumcontentistoolow,butourmediasupportsthegrowthofSp2/0andestablishedhybridomacellsverywell.TheproblemhereappearstobetheCO2content.Therefore,highcelldensitymaintainstheCO2atahigherlevel,andmorehybridomassurvive.After4to5days,addcompletemediasupplementedwithHTtothewells.After1week,testthewellswithhybridomasagainsttheantigenbyanELISA.Expandpositivehybridomasinto24wellplates.YouMUSTusespleenfeedercellsandHTmedia.Ifmanywellsarepositive,hybridomasmaybecombinedintoexpansionwells.CELLSMUSTBERE-EXPANDEDEVERYTWOTOTHREEDAYS.Biochemistrybookrecommendsthathybridomasbeclonedimmediately,inordertopreventovergrowthofthedesiredcells,bynon-secretingcellsthatmaybepresent.Summary:Themouseisimmunizedsixtimes.Afterthespleenisremoved,thesplenocytesareobtainedandfusedwithmyelomacellsusingPEG.Thehybridsareplatedinto96welltissuecultureplatesandobservedforcolonygrowth.
