CharlesE.Krininger,IIIandPeterJ.HansenDept.ofAnimalSciences,UniversityofFlorida

ThisprotocolwasadaptedfromonepreparedbyAydinGuzelogluandWilliamThatcher.ThecelllinewaspreparedbyT.R.Hansen,UniversityofWyomingandisavailablefromATCC.

CULTUREMEDIUMIngredientsthesecatalognumbersrefertoproductsfromSigma;productsfromothersupplierscanalsobeusedPowderedHamsF-12(N6760)PowderedMEMD-valinemodification(M7395)Fetalbovineserum(F4135)Horseserum(H1138)Antibiotic-antimycotic(ABAM)(A9909)D-valine(V1255)Insulin(I5500)

Preparation1.Place1000mlofdoubledistilledwaterin4000mlbeaker.2.Add10.7gofF-12and9.62gofMEM,allowtodissolve3.Add400IUofinsulin4.Dissolve0.0683gofD-valinein1mlof0.5NNaOH5.Addthe1mlvalinetoculturemix6.Add3.37gofsodiumbicarbonate7.AdjustpHto7.38.Bringtotalvolumeupto1600mlwithdoubledistilledwater9.Filter4aliquotsof400mlinto500mlbottles10.Storemediumat4Cuntilneeded11.Tomakemediumcontainingserum(forculture),add50mlofFBS,50mlofhorseserumand5mlofABAMtoeach400mlbottleCompletemediumcanbestoredat4^oCfor2weeks.Mediummustbewarmedforaminimumoftwohourspriortouse.

THAWINGCELLS1.RemoveampulefromLN₂storagetankandplacein37^oCwaterbath2.Invertsampleevery30secondsandplacebackintowaterbathuntilsampleisthawed.3.PourcontentsofstoragevialintoT175cultureflask.4.Add50mlofcompletemediumandplacein37^oCincubatorfor24hours.5.Removeoldmediumandreplacewith50mloffreshmedium.6.Placebackinincubatoruntilcellsbecomeconfluent(normally2-4days).7.Oncecellsreach>90confluency,theyarereadytobetrypsinized.

TRYPSINIZATION/SPLITTING1.Removeoldmedium2.ForT175flasksadd20mloftrypsin;forT75flasksadd10mloftrypsin.3.Placebackinto37^oCfor10minandtapflaskgentlytodislodgecells.4.Ifcellsarenotcompletelytrypsined,addanother10mloftrypsinandrepeatstep3.5.Oncecellsaredislodged,add10mlofcompletemediumtostoptrypsinization.6.Pourcontentsinto50mlcentrifugetubeandcentrifugefor10minat1000RPM.7.Removeoldmedium,beingcarefulnottodislodgepellet.8.ResUSPendpelletincomplete/serummedium:4mlforT175flaskand2mlforT75flask9.Transfer1mlcells/mediumtoT75flaskor2mlcells/mediumtoT175flask.10.Add30mlcomplete/serummediumtoT75flaskor50mltoT175flask.11.Placein37^oCuntilconfluency12.Repeattrypsinaztion/splittingprocedure

FREEZING1.Performtrypinizationproceduretostep8.2.Usinghemacytometer,calculatecellnumber.3.Dilutecellstoconcentrationof2millioncellspermlofmedium.4.Slowlydripinequalvolumeof20%(v/v)DMSOsolutioninculturemedium5.Allocatecellsin1mlcryotubes.Oneachtubemakenoteofdatenameandpassagenumber.6.Placeat-70^oCfor24hours.7.StorecellsinLN_2.8.Recordstoragelocation,passagenumber,date,andnameofpersonfreezingcellsinthecellbanklog.9.Thawandtest1sampleperfreezingruntotestviABIlity.

ReferencesusingBENDcellsJohnsonetal.Endocrine10:243(1999)Perryetal.Mol.Endocrinol.13:1197(1999)Austinetal.Endocrinology140:542(1999)

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