I.Purpose:

A.Samplesofsolidtumorsorlymphnodesmaybesentfrompatientswithcancer.Thesesamplesshouldbeprocesseddirectlyandalsosetupforshorttermcultureoftumorcells.Caremustbetakentoavoidover-growthofnormalfibroblastcellsthatarealsopresentinthetumorspecimen.

II.CultureProcedure:

A.Aseptictechniquemustbeusedwhensettingupthecultures,preferablyunderalaminarflowhood.Labela15mlcentrifugetubeforthedirectharvest,and3T-25flasksand1100mmpetridishforshorttermcultureswiththepatientnumber,patientname,typeoftissue,anddate.Flasksshouldalsobelabeled(usinggreekletters)toidentifyprimarycultureswhenflasksaresub-cultured;thisallowsforidentificationofculturalartifactsandpsuedovsrealmosaicism.B.CulturesaregrownincompleteNutrientMixF-10.Mediashouldbefresh(lessthan4daysold)andbeprewarmedandatpH7.0-7.5.Allculturesareincubatedinawet,5%CO2incubatorat37C.C.Usingforcepstransfertumortothepetridish,ifthesampleisverylargecutoffasection.Add3mlofmediatothedish.Usingscissors,cuttumorintopiecesabout5mmX5mm.Holdingacornerofthetumorwithforceps,mashitusingtheendofasterile5mlpipettofreethetumorcellsfromthetissue.Masheachpieceoftumorthoroughly.Transfer1mlofcellsUSPensiontothelabeledtubefordirectharvest,and1mltoeachof2T-25flasksforshorttermculture(1shouldbeharvestedafter24hours).Add2mlfreshmediatothetissueinthepetridish.Usingscissorscutthetissueintosmallbits.Transfer1mltothelastflaskforshorttermculture,leavingtherestinthepetridish.Add2mloffreshmediatoeachoftheflasksandthecentrifugetube,add4mlofmediatothepetridish.Incubatetheflaskscheckingforgrowthafterthethirdday.Tumorcellstendtogrowasnon-adherentcellsalthoughtheymaygrowincloseassociationwithfibroblasts.CulturesshouldbeharvestedassoonaspossIBLetominimizetheoccuranceofin-vitrochromosomeabnormalitiesthatmaynotreflectthetruekaryotypeofthetumor.Culturesarepassagedbyaddinganequalvolumeoffreshmediatotheflask.Whenthevolumebecomeslarge,transferhalftoanewflask.Assoonastheculturesaregrowingwelltheyshouldbeharvested.Transferhalftheculturetoanewflask,addanequalvolumeoffreshmediaandharvestthenextday.

III.HarvestProcedure:

A.Add30mclofcolcemidworkingsolutiontothetubeorflask.Incubatefor45minutes.Transfercellsuspensiontoa15mlcentrifugetubeifnecessary.B.Centrifugethetubesfor6minutesat150xg(900rpmintheSorvallGLC-2Bcentrifuge).C.Aspirateanddiscardallbut0.3mlofsupernatant.Resuspendthecellpelletbymixingbyhand,donotuseamechanicalvortexerasthismaybreakthecells.Add3mlofprewarmedhypotonicsolutionandmix.Incubatethetubesat37Cfor8minutes.D.Add2dropsoffixtothetubes.Centrifugethetubesfor6minutesat150xg.E.Aspirateanddiscardallbut0.3mlofsupernatant.Mixbyhandtobreakupthecellpellet.F.Slowlyadd2mloffixative(1pasteurpipetful)lettingitrundownthesideofthetubesothatitlayersontopofthecellsuspension.Mixrapidlybyhandsothatallthecellsuspensionisfixedevenly.Washdownsidesoftubewithanotherpipetfuloffix.Letsitatroomtemperaturefor15-20minutes.G.Centrifugefor6minutesat150xg.H.Aspirateanddiscardallbut0.3mlofsupernatant.Resuspendthecellpellet.Add2mloffixativeandmix,washdownsidesofthetubewith2mlfix.Letsitatroomtemperature15-20minutes.I.RepeatstepsG,H1or2timesuntilthepelletiscleanandwhite.Thecultureisnowreadytomakeslides.Forbestresults,slidesshouldbemadethesamedayastheharvest.

IV.SlidePreparation:

A.Seeprocedureforregularlymphocytecultures.B.Ifpossible,stainoneslidewithouttrypsinizationinordertocheckforthepresenceofdmins.

V.Solutions:

Colcemidworkingsolution:10mgColcemidin250mldH2O,sterilefiltered,storeat4C.CompleteNutrientMixF-10:100mlNutrientMixF-10,20mlFetalcalfserum,1mlPenicillin/Streptomycinsolution,1mlL-Glutaminesolution,storeat4C.Fixative:30mlMethanoland10mlGlacialAceticAcid,preparedfresh.Hypotonicsolution0.075MKCl:2.8gPotassiumchloridedissolvedin500mldH2O.

VI.Reagents:

Colcemid:Gibcocat#890-3014.10mgbottle.Fetalcalfserum:HYCLONE/STERILESYSTEMSINC.cat#A-1111-D.definedfetalbovineserum100mlbottle,storefrozen.GlacialAceticacid:AMERICANSCIENTIFICPRODUCTScat#9508-2.Aceticacid,GlacialACS(Aldehydefree),500mlbottle.L-Glutamine:GIBCOcat#320-5030.L-Glutaminesolution100X(200mM),20mlbottle,storefrozen.Methanol:AMERICANSCIENTIFICPRODUCTScat#3016-1.MethylalcoholanhydrousAR(ACS)(Absolute)Acetonefree,500mlbottle.NutrientMixF-10:GIBCOcat#430-1200.NutrientMixtureF-10(HAM)powderedform,preparedtoinstructions,10x1literpackage.Penicillin/Streptomycin:GIBCOcat#600-5140.Penicillin/Streptomycinsolution,10,000units/ml/10,000mcg/ml,20mlbottle,storefrozen.PotassiumChloride:COLUMBUSCHEMICALINDUSTRIES,INC.ACSgranular,500glots.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。