Materials

1. DMEM,highglucose(LifeTechnologies,Inc.#10313-021orequivalent)
2. FetalBovineSerum(LifeTechnologies,Inc.#16000-044orequivalent)
3. L-glutamine(LifeTechnologies,Inc.#25030-149orequivalent)
4. HAT(LifeTechnologies,#31062-0211orequivalent)
5. HybridomaCloningFactor(Fisher#IG50-0615)
6. PEG1500(BoehringerMannheim#0783-641orequivalent)
7. Scissors
8. Forceps
9. 50mlsterilecentrifugetubes(Falcon#2070)
10. 96wellcultureplates(Falcon#3072))
11. 6-wellcultureplates(Falcon#3046)
12. Hemocytometer
13. TrypanBlue,0.4%(LifeTechnologies,Inc.#630-5150AG)
14. Multi-channelpipettorandsteriletips
15. ReagentReservoir
16. HT(LifeTechnologies,Inc.#11067-030)
17. Petridishes(Falcon#1009)
18. Ethanol,70%
19. PBS,Sterile(LifeTechnologies,Inc.#20012-027)

Procedure

1. PreparationofSpleencells
   1. SacrificetheanimalandswabtheaBDominalareainalcohol.
   2. Opentheabdominalareaandlocatethespleen.
   3. Usingsterileforcepsandscissors,removethespleenandplaceinatubecontaining50mlsterilePBS.
   4. TransferaspleentoaPetridishcontaining50mlsterilePBS.Removeanyexcesstissueandfat.
   5. Washthespleenbytransferringtoa6-wellplatecontaining5ml/wellsterilePBS.
   6. TransferthespleentoaPetridishcontaining50mlsterilePBS.
   7. PrepareasinglecellsUSPensionbyteasingthetissuewithsterileforceps.
   8. Collectthecellsintoa50mltube.WashthePetridishwithanadditional10mlsterilePBSandaddtothetube.
   9. Allowthecellstosettlefor1minutes.
   10. Carefullyremovethecellsuspensionandtransfertoacleantube,beingcarefulnottodisturbthelargerpiecesoftissueatthebottomofthetube.
   11. Washthetubewith10mlsterilePBSandallowtosettlefor1minutebeforetransferringandcombiningwiththeremainingcellsuspensioninthetube.
   12. Centrifugeat1000rpm,5minutes,roomtemperature.
   13. Carefullysiphonoffthesupernatantanddiscard.Tapthepellettoresuspend.Washthecellswith50mlsterilePBS.
   14. Carefullysiphonoffthesupernatantanddiscard.Resuspendthecellsin10mlsterilePBS.
   15. TakeanaliquotforacellandviABIlitycount.
2. PreparationofMyelomaCells.
   1. Collectmyelomacellsfrom2-4T-150flasksinlogphaseofgrowth.
   2. WashtwicewithsterilePBS.
   3. Resuspendthecellsin10mlsterilePBS.
   4. Takeanaliquotforacellandviabilitycount.
3. FusionProcedure.
   1. Mixthespleenandmyelomacellstogetherina50mltubeataratioof2:1-5:1(Spleen:myeloma).TopoffwithPBS.
   2. Centrifugeat800rpmfor5minutes,roomtemperature.
   3. Aspirateallofthesupernatant.Tapthebottomofthetubetoloosenthepellet.
   4. SlowlyaddthePEG,dropwiseoveraminute,usingthepipettostirthecells.
   5. Continuemixingforanother60seconds.
   6. DilutethePEGwithsterilePBS:1mlin1minute5mlin1minute10mlin1minute.
   7. TopsoffthetubewithsterilePBS.
   8. Centrifugeat800rpm,5minutes,RT.
   9. Discardthesupernatant.ResuspendthepelletinHATmediumto5x10⁵cells/ml.
   10. Plate200ml/wellto96wellplates.Incubateat37^oC,8-10%CO₂for5-7days.