Materials

DMEM,highglucose(LifeTechnologies,Inc.#10313-021orequivalent)

FetalBovineSerum(LifeTechnologies,Inc.#16000-044orequivalent)

L-glutamine(LifeTechnologies,Inc.#25030-149orequivalent)

HybridomaCloningFactor(Fisher#IG50-0615)

50mlsterilecentrifugetubes(Falcon#2070)

15mlsterilecentrifugetubes(Falcon#2099)

96wellcultureplates(Falcon#3072))

Hemocytometer

TrypanBlue,0.4%(LifeTechnologies,Inc.#630-5150AG)

Multi-channelpipettorandsteriletips

ReagentReservoir

HT(LifeTechnologies,Inc.#11067-030)

Procedure

Thedaybeforethecloning,refeed24-wellplatesorflaskswithfreshmedium.

Preparethecloningmedium

DMEM

4mML-glutamine

20%FBS

10%HybridomaCloningFactor

ResUSPendthecellstobecloned.Transfer1mltoasterile15mltube.Transfer50mlofthissuspensiontoacleantubeforcellandviABIlitycounts.

Countthecellsanddeterminetheviability.NOTE:Theviabilitymustbegreaterthan80%tocontinue.

Foreachhybridomacellline,calculatethedilutionstogive4cells/ml,2cells/mland1cell/mlincloningmedium.

Label50mltubesforeachcloneanddilution.

Addmediumtoeachtubeaccordingtothecalculateddilutions.

Seriallydiluteeachcloneto4,2,and1cell/ml.Thefinaldilutiontubeshouldcontain50mlofcloningmediumat1cell/ml.

Poureachofthedilutionsintoasterilereagentreservoir.Plate250ml/wellinto96-wellplates-1plateat4cells/ml,1plateat2cells/mland2platesat1cell/ml.

Completedilutionsandplatingforeachhybridomacellline.

Placeallplatesat37oC,8-10%CO2.Incubatefor5-7days.

Microscopicallyexamineallplatestoensurecloningandplatingefficiencybeforerefeedingtheplates.