Iftitreispositivedooneofthefollowing2weekslater:

1. Boosttailveinwith20ug-50ugofAntigeninPBSandproceedwithfusiononday4.OR
2. Boostsubcutaneouslywith50ug-100ugofAntigeninPBSandproceedwithfusion4dayslater.OR
3. Boost3daysinarowwith15ugAginPBSandproceedwithfusionon4thday.

CellLines:

1. Myeloma;P3X63-Ag8.653

   -	Origin:BALB/c,nonsecreting,8-azaguanineresistant,HPRT-.

2. Myelomafox-NY

   -	Origin:Robertsonian,8-azaguanineresistant,HPRT-,APRT-.
   -	(micehaveresistancetodrugandexpressionofheavychainonthesamechromosome).

3. Macrophage-derivedJ774A.1

Maintenanceofthecells:

Stocksolutions:

Seedmacrophagesatadensityof1.5x105cells/mlinthemediumdescribedonnextpage.Add2.5ug/mlLPSwhichinducesdifferentiation.

Collectsupafter2-3days,orwhenmediumisgettingtooyellow.Induce2moretimes,eachtimewith1ug/mlLPSandcollectsupafter2dayseach.

Poolthesups,filteranduseasrecommended.(Couldbealiquotedandstoredat-20C.)

PreparationofMedia:

forP3X63-Ag8.653:IMDMcompleteto425mlofIMDMadd:

forfox-NY:IMDMorRPMI

forJ774A.1:samemediumastheonefortheP3X63-Ag8.653(=Ag8).

Growthconditions:

TheMacrophagesoptimaldensityis1.5x105/ml.Whenexpandingthem,usea"policeman"toscrapethem;thisiseasiertodoiftheyaregrowinginpetridishesatthisstage.

FreezingHybridoma/Myeloma/macrophages.

Freezingsolution:90FCS+10DMSO,icecold.

1. Spindown107cells(106minimum)at1200rpmfor5min.
2. Aspiratemedium.
3. ResUSPendin1mloficecoldfreezingsolution.
4. Transfervialtoaninsulatedfreezingboxandplaceat-70°Cforatleast1hr.(couldbeforacoupleofdays).
5. Transferthevialfromthe-70°Ctotheliquidnitrogentankandlogtheentryinthefreezerlog.

Thawingcells:

1. Takevialoutofliquidnitrogentankandthawitimmediatelyina37°Cbath(about1min).
2. Whenthereisstillasmallpieceoficeleft,dilutethecellsbytransferringthemintoaconicaltubecontaining10mlofthegrowthmediumat37°C.
3. Spinat1200rpmfor5min.
4. Aspiratemediumandresuspendcellsin5mlofmedium,ina25cm2flask.

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