MouseImmunization
Order6sixweekoldBalb/CmiceandlettheARCknowtheyarecoming.Haveyourantigenreadyforwhentheyarrive.Oncetheygetthereearmarkthemiceandperformapre-bleedonthemtobeusedasanELISAcontrolformonitoringthetiterandscreeningofthehybridomas:
BleedingMice
- Placethemouseinamouserestrainer.
- Sterilizethetailwith70ethanol.
- Witharazorblade,nipoffthelast2mmofthetipofthetail.
- Usingamilkingmotion,pullblooddownandletdripofftheendofthetailuntilyouhavecollected~200µL.(Youmayhavetopre-bleedtwice,withaweekorsobetweenbleeds).
- Takethecollectedbloodandplaceat37°Cfor30min.toremovecomplement.
- Placebloodat4°Covernighttoclot.
- Centrifugesamplesat10,000g10min.
- Pipetofftheserumsupernatant.Storeat-20°C.Thisisyourpre-bleedcontrol.
HybridomaFusion
Foronefusionyouwillneed:
- 8500mLbottlesofDMEMmedia
- 3500mLbottlesoffetalcalfserum(FCS)
- 250mLbottlesof100Xpenicillin-streptomycin
- 10mLof100XHATselectionsolution
- 10mLof100XHTsolution
- 10sterileflat-bottomed96-wellplates
AssayingforPositiveClones
RunanindirectELISAusingtheantigenyouwanttheMAbdirectedagainst.IfyouareraisingtheMAbsagainstasmallhaptenthatyoucoupledtoacarrierproteinforimmunizationofthemice,thenusethehaptencoupledtoadifferentcarrierproteinforthescreen.
ExpandingPositiveClones
- Thedaypriortoanyexpansion,obtainfeedercells.Forthisfirstexpansionfromthe200µLculturesin96-wellplatesto500µLculturesin24-wellplates,add200µLoffeedercells/wellinDMEM/20FCS+HTtothe24-wellplatesthedaybeforeexpansion.LetthefeedercellsgrowovernightintheCO2incubator.
- Fortheinitialexpansion,resUSPendthepositivetestinghybridomacellsandplaceallbutthelast10µL(200µL)inthewellofthe24-wellplatewiththefeedercells.Thenbringto500µLwithDMEM/20FCS+HT.
- Add200µLofDMEM/20FCS+HTtothelittlebitofcellsleftinthe96-wellplate.Thisservesasabackupforyourpositiveclones.
- PlaceallplatesintheCO2incubator.
- Whenthecellsarebeginningtoacidifythemedia(getsorange-yellow),changethemediaanddoubletheculturevolumebypipettingoff400µL,andreplacewith900µLfreshDMEM/20FCS+HT.
- Harvestmorefeedercellsforyournextexpansion.Put0.5mLffeedercellsinDMEM/20FCS(notetheremovaloftheHT)inaT25flask(oneforeachpositiveclone)forlateruse
- Whenthecellsarereadyforexpansionoutofthe24-wellplate,resuspendthemandpipettotheT25flaskswithfeedercells.Againleavealittlebitinthe24-wellplateandrefillwithDMEM/20FCSasabackup.VolumetotheT25culture5mLwithDMEM/20FCS(add3.5mL)Placeallculturesintheincubator.
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