MNinHumanLymphocytes(methoddescription)LymphocyteisolationLymphocyteswereisolatedusingFicoll-PaquedensitygrADIents.Bloodwasdiluted1:1withphosphatebufferedsaline,andlayeredontoFicoll-Paquewithratioofblood+PBS:Ficollmaintained4:3.Thebloodwascentrifugatedat1800rpmfor35minatroomtemperature.Thelymphocytelayer(buffycoat)wasremovedandwashedtwiceinPBSat1200rpmfor10mineach,followingthesamewashwithmediaRPMI1640.Celldensitywascountedwithahemocytometer.CultureconditionsLymphocyteswereculturedat37oinahumidified,5%CO2incubatorin15mlconicalpolystyrenecentrifugetubes.Typically,eachcultureconsistedofaninitialdensityof1x106cellsin2mlsofculturemedium.CulturemediumconsistedofRPMI1640supplementedwith10%fetalbovineserum2mML-glutamine,100units/mlpenicillin,100ug/mlstreptomycinand1.5%phytohemagglutinin.MicronucleusassayCytochalasinBwasaddedtotheculturesat44hourspostinitiationasdescribedbyFenechandMorley(1985).CytochalasinBpreventsthecellsfromcompletingcytokinesisresultingintheformationofmultinucleatedcells.FinalconcentrationofcytochalasinBinthecellcultureswas3mg/ml.Lymphocyteswerespundirectlyontoglassslidesat72hrsaftercultureinitiationusingacytocentrifuge(Shandon,Sewickley,PA).Thecellswouldthenbespunontotheslidefor10minat600rpm.Slideswouldbeallowedtoairdrybeforemethanolfixationatroomtemperaturefor15min.Slideswerestoredat-20oCinasealedbox,desiccated,underN2.Detailedproceduresforperformingtheantikinetochoreantibodystainingmethodhavebeendescribedelsewhere(EastmondandTucker,1989;Yageretal.,1990).Briefly,methanolfixedslidesareincubatedfor5mininPBScontaining0.1%Tween20.Excessfluidisdrainedfromtheslideand40-50uloftheantikinetochoreantibody(Chemicon,LosAngeles,CA)diluted1:1withPBScontaining0.2%Tween20isapplied.Theslideisthencoverslippedandplacedinahumidifiedchamberat37oCfor1h.FollowingtwowashesinPBScontaining0.1%Tween20for5mineach,excessfluidisagaindrainedandtheslidesarecoveredwith1:50dilutionoffluorescentgoatanti-humanIgG(Chemacon,LosAngeles,CA)andincubatedagainfor1h.Becausethefluorescent-labeledantibodieswillfadeuponexposuretolight,thisandallsubsequentstepsshouldbeconductedinyellowlight.Theslideswererinsedtwiceinbufferplus0.1%Tween20andcounterstainedwith4"6-diamidino-2-phenylindole(DAPI)(2ug/ml)inanantifadesolution(JohnsonandNogueiraAraujo,1981).Forthemicronucleustest,1000binucleatelymphocytes(thosethathaveundergoneonemitoticdivision)werescored,500cellsfromeachduplicateculture,forthenumberofmicronuclei.Thepresenceorabsenceofakinetochorespotwasdeterminedbyswitchingtothefluorescentfilter.Scoringcriteriawereasfollows:1)cellsshouldhavearoundorovalappearancewithanintactcytoplasm,2)nucleishouldsimilarlyberoundorovalwithanintactnuclearmembrane,3)onlycellshavingundergoneonenucleardivisionshouldbescoredforthepresenceofmicronuclei,4)micronucleishouldbecountedonlyiftheyareonethirdorlessthesizeofthemainnuclei,5)micronucleishouldbestainedsimilartothemainnucleiand6)micronucleishouldbeclearlyseparatedfromthemainnuclei.Scoringwasperformedbytwoscorerswith10%ofslidesbeingrescored.Nodifferenceinscoringwasfound.AllquestionablemicronucleiwereadditionallyassessedbythirdCytogeneticsist.Replicativeindex(RI),ameasureofcelldivisionkinetics,wasscoredfromthesameslidesscoredformicronucleibycountingthepercentofcellscontaining1,2,3ormorenucleioutof400totalcellsperindividual.RIwascalculatedasfollows:RI=(1x%mononuclearcells)+(2x%binuclearcells)+(3x%tri)+(4x%tetra）/100