MouseImmunization

Order6sixweekoldBalb/CmiceandlettheARCknowtheyarecoming.Haveyourantigenreadyforwhentheyarrive.Oncetheygetthereearmarkthemiceandperformapre-bleedonthemtobeusedasanELISAcontrolformonitoringthetiterandscreeningofthehybridomas:

BleedingMice

1. Placethemouseinamouserestrainer.
2. Sterilizethetailwith70ethanol.
3. Witharazorblade,nipoffthelast2mmofthetipofthetail.
4. Usingamilkingmotion,pullblooddownandletdripofftheendofthetailuntilyouhavecollected~200µL.(Youmayhavetopre-bleedtwice,withaweekorsobetweenbleeds).
5. Takethecollectedbloodandplaceat37°Cfor30min.toremovecomplement.
6. Placebloodat4°Covernighttoclot.
7. Centrifugesamplesat10,000g10min.
8. Pipetofftheserumsupernatant.Storeat-20°C.Thisisyourpre-bleedcontrol.

HybridomaFusion

Foronefusionyouwillneed:

1. 8500mLbottlesofDMEMmedia
2. 3500mLbottlesoffetalcalfserum(FCS)
3. 250mLbottlesof100Xpenicillin-streptomycin
4. 10mLof100XHATselectionsolution
5. 10mLof100XHTsolution
6. 10sterileflat-bottomed96-wellplates

AssayingforPositiveClones

RunanindirectELISAusingtheantigenyouwanttheMAbdirectedagainst.IfyouareraisingtheMAbsagainstasmallhaptenthatyoucoupledtoacarrierproteinforimmunizationofthemice,thenusethehaptencoupledtoadifferentcarrierproteinforthescreen.

ExpandingPositiveClones

1. Thedaypriortoanyexpansion,obtainfeedercells.Forthisfirstexpansionfromthe200µLculturesin96-wellplatesto500µLculturesin24-wellplates,add200µLoffeedercells/wellinDMEM/20FCS+HTtothe24-wellplatesthedaybeforeexpansion.LetthefeedercellsgrowovernightintheCO2incubator.
2. Fortheinitialexpansion,resUSPendthepositivetestinghybridomacellsandplaceallbutthelast10µL(200µL)inthewellofthe24-wellplatewiththefeedercells.Thenbringto500µLwithDMEM/20FCS+HT.
3. Add200µLofDMEM/20FCS+HTtothelittlebitofcellsleftinthe96-wellplate.Thisservesasabackupforyourpositiveclones.
4. PlaceallplatesintheCO2incubator.
5. Whenthecellsarebeginningtoacidifythemedia(getsorange-yellow),changethemediaanddoubletheculturevolumebypipettingoff400µL,andreplacewith900µLfreshDMEM/20FCS+HT.
6. Harvestmorefeedercellsforyournextexpansion.Put0.5mLffeedercellsinDMEM/20FCS(notetheremovaloftheHT)inaT25flask(oneforeachpositiveclone)forlateruse
7. Whenthecellsarereadyforexpansionoutofthe24-wellplate,resuspendthemandpipettotheT25flaskswithfeedercells.Againleavealittlebitinthe24-wellplateandrefillwithDMEM/20FCSasabackup.VolumetotheT25culture5mLwithDMEM/20FCS(add3.5mL)Placeallculturesintheincubator.