1. DMEM,highglucose(LifeTechnologies,Inc.#10313-021orequivalent)
2. FetalBovineSerum(LifeTechnologies,Inc.#16000-044orequivalent)
3. L-glutamine(LifeTechnologies,Inc.#25030-149orequivalent)
4. HybridomaCloningFactor(Fisher#IG50-0615)
5. 50mlsterilecentrifugetubes(Falcon#2070)
6. 15mlsterilecentrifugetubes(Falcon#2099)
7. 96wellcultureplates(Falcon#3072))
8. Hemocytometer
9. TrypanBlue,0.4%(LifeTechnologies,Inc.#630-5150AG)
10. Multi-channelpipettorandsteriletips
11. ReagentReservoir
12. HT(LifeTechnologies,Inc.#11067-030)

Procedure

1. Thedaybeforethecloning,refeed24-wellplatesorflaskswithfreshmedium.
2. PreparethecloningmediumDMEM4mML-glutamine20%FBS10%HybridomaCloningFactor
3. ResUSPendthecellstobecloned.Transfer1mltoasterile15mltube.Transfer50mlofthissuspensiontoacleantubeforcellandviABIlitycounts.
4. Countthecellsanddeterminetheviability.NOTE:Theviabilitymustbegreaterthan80%tocontinue.
5. Foreachhybridomacellline,calculatethedilutionstogive4cells/ml,2cells/mland1cell/mlincloningmedium.
6. Label50mltubesforeachcloneanddilution.
7. Addmediumtoeachtubeaccordingtothecalculateddilutions.
8. Seriallydiluteeachcloneto4,2,and1cell/ml.Thefinaldilutiontubeshouldcontain50mlofcloningmediumat1cell/ml.
9. Poureachofthedilutionsintoasterilereagentreservoir.Plate250ml/wellinto96-wellplates-1plateat4cells/ml,1plateat2cells/mland2platesat1cell/ml.
10. Completedilutionsandplatingforeachhybridomacellline.
11. Placeallplatesat37^oC,8-10%CO₂.Incubatefor5-7days.
12. Microscopicallyexamineallplatestoensurecloningandplatingefficiencybeforerefeedingtheplates.