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蚂蚁淘在线 / 品牌中心 / Ursa BioScience品牌新闻

Fusion and Cloning

作者： 时间：2026-07-18 点击量：

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,sterile
  • 100mmsterilePetriDishes(Falcon#1009)
  • 96-wellculturedishes(Falcon#3072)
  • 24-wellculturedishes(Falcon#3047)
  • Forceps
  • Scissors
  • Multi-channelpipettor,50-200ml
  • Pipettips,sterile
  • ReagentReservoir,sterile
  • Tupperwarecontainer

    • MyelomaCells

  • 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.1mlover1minute3mlover1minute10mlover1minute
  • Incubatefor5minutesinawaterbathat37oC.
  • Slowlyadd40mlofMediumA.
  • Centrifugethecellsat400gfor7minutes.
  • Discardthesupernatantandwashthecellin40mlofMediumA.
  • Slowlyresuspendthepelletin10mlofMediumC.
  • TransfertoaT75flaskcontaining40mlofMediumC.
  • Incubate16-24hoursat37oC,5CO2.
  • ThawMediumDandmix.
  • Transferthecellsfromtheflaskinto2x50mlcentrifugetubesandcentrifugeat400gfor10minutes.
  • Discardthesupernatantsandtaptoloosenthepellets.
  • CombinethepelletsandtransferthecellstoMediumD.Mixgentlybyswirlingthetube.
  • Letsitfor30minutesat37oC,5CO2.
  • Plate9.5mlofcellsinto10-100mmPetridishes.Tilttheplatestolevelthemixture.
  • TransfertheplatestoaTupperwarecontainercontainingaPetridishwith10mlsterilewater.
  • Incubateplatesat37oC,5CO2.
    • Maintenance
  • After10-14days,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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