Materials:P3X63Ag8.653murinemyelomaorYB2/0(maintainedat<1x10⁶/ml)50%w/vPEG1500,warmedto37°C

Medium:IMDMsupplementedwith20%fetalbovineserum,4mML-glutamine,1mMsodiumpyruvate,50Upenicillin,50µgstreptomycinand50µM2-MEintheabsenceorpresenceofHATorHTforselection(I-20).

Procedure:

NOTE:Allwashesareat1,000rpmfor10minutesat4°Cusingserumfreemedia

TissueCollection:1.SacrificeanimalbyCO₂inhalation.2.Removeperitonealcellsfromnaivemouseforuseasfeedercellsbyperitoneallavage.PlacecellsonICE.3.Sacrificeimmuneanimalandremovespleen.PlaceonICE.

CellPreparation:1.Teasespleeninicecoldserum-freemedium(I-0).PasscellsUSPensionthroughaFalcon70microncellfilterandsuspendin50mloficecoldI-0.Centrifugeandwashcellsthreetimesat4°CinI-0.Resuspendcellsafterthethirdwashin10mlI-0andcountviablecells.Keepcellsonice.2.Concurrentlywiththespleenocytes,centrifugeandwashmyelomacellsthreetimes,usingI-0andresuspendinI-0.Countviablecells.3.Inaddition,washperitonealcellsinI-0twice,resuspendinI-20andcount.4.Addanappropriatenumberofmyelomacellstotheentirevolumeofspleencellsaccordingtothefollowingratiosandcentrifugetogether.

Species	Spleenocytes:Myeloma
mouse-mouse	5:1
hamster-mouse	4:1
rat-mouse	5:1
Tcellfusion	1:1

FusionProtocol:1.Aspirateallsupernatantandsuspendpelletbytappingtheendofthetube.Placetubeincontainerofwarmwater(37°C).Gradually,overaperiodof30seconds,add1mlof37°CPEGwhiletappingthesidefothetubetoachievethoroughmixing.Overthenext90secondscontinuetomix.Afterapproximately1minute40secondsstopmixingandfilla5mlpipetwithwarmI-0.Whenexactly2minuteshaveelasped,dilutethePEG/cellmixtureslowlybyaddingdropwise1mlofI-0overa1minutetimespan.Duringthenext1minute,add2mlI-0dropwise.Theremaining2mlI-0inthepipetisaddedduringthenext40seconds.Nextusea10mlpipetandadd14ml37°CI-0duringthelast1minuteperiod.Bringthetotalvolumeto50mlusingI-20.Centrifugeat4°CandresuspendinHATmediumattheappropriatevolumetobringthecellstothefollowingconcentrations:

Type	CellConcentration
mouseorrat	1.5x106cellsperml
hamster	0.5-1.0x106cellsperml

Alternatively,thecellscanberesuspendedinI-20+2-MEwith150µladdedperwell.Anadditional50µlofa4XHATsolutioncanbeaddedat16-24hoursafterfusion.

2.Addperitonealcellstothefusedcellsat2.5x10⁴cellsperml(thiswillresultinaplatingof5x10³PECperwell).

3.Dispensecellsinto96wellplatesasfollows:CellsinI-20+2-ME150µlperwell+50µlperwell4XHATafter18-24hrs

FeedingSchedule:

Thedayofthefusionisconsideredday0.Fusionplatesareexaminedat24-48hoursforanyabnormalities(i.e.bacterialcontamination).Onday7,wellsareinspectedvisuallyandthenfed.Onehalfofthevolumeineachwellisaspiratedusingasterilepasteurpipet.Anewpipetisusedforeachplate.Wellsarefedwith125µlofI-20+2ME+HATondays7,11andthereafterasneeded,i.e.Mon.,Wed.,Fri.

Culturesareexaminedvisuallyateachfeeding.Onceamajorityofwellsappear50%confluentforgrowth,supernatantsareharvestedforscreeningbytheinvestigator.Platesarefedatthistime.

**Investigatorsshouldprovideinformationontheserumtiteroftheimmunizedanimal.Ifthetiterisgreaterthan1:10,000,itmaybeimportanttofeedtheculturesatleast3-4timespriortothescreening.Thiswillessentially"washaway"anyantibodyreleasefromdyingBcellsanddecreasebackgroundIglevels.

Kohler,G.andC.Milstein.1975.Continuousculturesoffusedcellssecretingantibodyofpredefinedspecificity.Nature256:495.

Sanchez-Madrid,F.,P.SzklutandT.A.Springer.1983.Stablehamster-mousehybridomasproducingIgGandIgMhamstermonoclonalantibodiesofdefinedspecificity.J.Immunol.130:309.

Sheehan,K.C.F.,J.CalderonandR.D.Schreiber.1988.GenerationandcharacterizationofmonoclonalantibodiesspecificforthehumanIFNgammareceptor.J.Immunol.140:4231.

Sheehan,K.C.F.,N.H.RuddleandR.D.Schreiber.1989.GenerationandcharacterizationofhamstermonoclonalantibodiesthatneutralizemurineTumorNecrosisFactors.J.Immunol.142:3884.

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