Reagents(StemCellTechnologies,Inc.#03800)MediumA-Pre-fusionMediumandHybridomaExpansionMedium(StemCellTechnologies,Inc.-#03801)MediumB-FusionMedium(StemCellTechnologies,Inc.-#03802)MediumC-HybridomaRecoveryMedium(StemCellTechnologies,Inc.-#03803)MediumD-HybridomaSelectionMedium(StemCellTechnologies,Inc.-#03804)MediumE-HybridomaGrowthMedium(StemCellTechnologies,Inc.-#03805)PEGSolution(StemCellTechnologies,Inc.-#03806)Materials
50mlSterileconicaltubes(Falcon#2070)15mlSterileconicaltubes(Falcon#2099)10mlsterilepipets(Falcon#7551)1mlsterilepipets(Falcon#7521)PasteurPipets,sterile100mmsterilePetriDishes(Falcon#1009)96-wellculturedishes(Falcon#3072)24-wellculturedishes(Falcon#3047)ForcepsScissorsMulti-channelpipettor,50-200mlPipettips,sterileReagentReservoir,sterileTupperwarecontainerMyelomaCells
Oneweekpriortothefusion,splitmyelomacellsintoMediumAtoensurethattheyarewelladapted.Growupapproximately2x107healthycells,inmid-logphase,foreachfusion.Fusion
CountthemyelomacellsandresUSPendto2x107cellsin30mlMediumAina50mltube.Sacrificedthemouse,saturateinethanol,andremovethespleen.PlacethespleeninaPetridishcontaining10mlofMediumA.Prepareasinglecellsuspensionofthespleen.UsingaPasteurPipet,transferthespleencellstoa50mltube.RinsethePetridishwithanother10mlofMediumAandaddtothetube.Allowthetubetositforapproximately1minutetosettlethelargerpiecesoftissue.Transferthecellsuspensiontoacleantube,leavingbehindthelargerpiecesoftissue.Add10mlofMediumAtothetubetowashthetissuepieces.Allowtosettle.Transferthemediumtothecleantube,combiningitwiththepreviouscellsuspension.Centrifugethesplenocytesuspensionat400gfor10minutes,RT.Resuspendthecellsin10mlofMediumAandcount.Combine108viablespleencellswith2x107myelomacellsina50mltube.Centrifugeat400gfor10minutes.Discardthesupernatantandwashthepellettwicewith40mlMediumB,pre-warmedto37oC.Discardthesupernatant.Tapthebottomofthetubetoloosenthepellet.Add1mlofPEGsolutiontothepelletovera1minuteperiod,continuallystirringthecells.Continuestirringforanadditional1minutes.StopthefusionbyaddingMediumBwhileconstantlystirring.1mlover1minute3mlover1minute10mlover1minuteIncubatefor5minutesinawaterbathat37oC.Slowlyadd40mlofMediumA.Centrifugethecellsat400gfor7minutes.Discardthesupernatantandwashthecellin40mlofMediumA.Slowlyresuspendthepelletin10mlofMediumC.TransfertoaT75flaskcontaining40mlofMediumC.Incubate16-24hoursat37oC,5CO2.ThawMediumDandmix.Transferthecellsfromtheflaskinto2x50mlcentrifugetubesandcentrifugeat400gfor10minutes.Discardthesupernatantsandtaptoloosenthepellets.CombinethepelletsandtransferthecellstoMediumD.Mixgentlybyswirlingthetube.Letsitfor30minutesat37oC,5CO2.Plate9.5mlofcellsinto10-100mmPetridishes.Tilttheplatestolevelthemixture.TransfertheplatestoaTupperwarecontainercontainingaPetridishwith10mlsterilewater.Incubateplatesat37oC,5CO2.MaintenanceAfter10-14days,examinetheplatesforthepresenceofcolonies.Removeisolatedcoloniesfromtheplatesusingapipettorwithasteriletips.Setthepipettorfor10ml.Pipeteachcolonyintoaseparatewellof96-wellplatescontain200mlofMediumE.Incubatetheplatesat37oC,5CO2for1-4dayswithoutfeeding.Removethesupernatantsfromthewellsandtest.Refeedthewellswith200mlMediumEorotherHybridomaexpansionmedium.RecloninginClonaCell-HYNote:Morethan95ofthecolonieswillbemonoclonalwhenselectedbyClonaCell-HY.RecloningcanbedonetoensurestABIlity.
Oncethecellsaregrowingwellin24-wellplates,resuspendthecellswitha1mlpipet.Remove10mlofthecellsuspensionandaddto1.0mlofMediumA.Mixwell.Remove100mlofthissuspensionandaddtoatubecontaining10mlofMediumD.Mixwellandaddtoa100mmPetridish.Spreadevenlybytiltingtheplates.Incubateat37oC,5CO2aspreviouslydescribed.Repeatforeachclone.After10-14days,selectcoloniesandtransferto96-wellplatesbeforetesting.
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