流式分析软件Flowjo使用说明

FlowJoBasicTutorialFlowJosoftwareisusedfortheanalysisofflowcytometrydata.ThistutorialwillintroduceyoutoFlowJousingasimpleexperimentasanexample.AFITCanti-CD8reagentisti-tratedtodeterminethemosteconomicalconcentrationforstaininginfutureexperiments.•FlowJoBasicTutorial••©MMVI-iii•TableofContentsIntroduction.....................................................................................................1Step1:Loadsamplesintotheworkspace.......................................................3Step2:Analyzethecontrolsampleindetail..................................................7Step3:Copygatesandstatisticstoallsamples..............................................12Step4:Verifygatesonallsamples.................................................................13Step5:Generateagraphicalreport................................................................17Step6:Generateatableofstatistics...............................................................23FlowJowaswrittenbyAdamTreisterandMarioRoederer,basedonconceptsdevelopedwithDavidParks,MartinBigosandWayneMoore.WeareindebtedtotheHerzenberglaboratoryatStanfordandouractiveandenthusiasticusersfortheirideas,discussionsandtirelesstestingofnewversions.FlowJoisCopyright©TrusteesofLelandStanfordJr.University,1996-97FlowJoisCopyright©TreeStar,Inc.1997-2006FlowJoforMacintoshisCopyright©TreeStar,Inc.2006FlowJoforWindowsisCopyright©TreeStar,Inc.2006FlowJoTutorialandWebSiteareCopyright©TreeStar,Inc.1997-2006FlowJoisaproductofTreeStar,Inc.[phone]International:[e-mail]tionFlowJosoftwareisusedfortheanalysisofflowcytometrydata.YoucanuseFlowJotoanalyzeallofyourflowcytometrydata-regardlessofthecytometerusedtocollectyourdatafiles.ThistutorialwillintroduceyoutoFlowJoandtothe6stepsinvolvedinanalyzingabasicimmunophenotypingexperiment.1.LoaddataintotheWorkspace.2.Analyzethecontroltubedataindetail(gatesubsetsandaddstatistics).3.CopygatesandstatisticstoAllSamples.4.VerifygatesonAllSamples.5.GenerateagraphicalreportincludingAllSamples.6.GenerateatableofstatisticsfromAllSamples.Instructionstoguideyouthroughtheanalysisofabasicimmunophenotypingexperi-mentarecontainedinthistutorial.FollowalongwiththeprovideddatafilestogetafeelforhowFlowJoworks.Ifyouwishtoskipsteps,weprovidepre-madeWorkspacefilesthatcorrespondtoeachanalysisstep.ThedemonstrationdataandWorkspacefilescanbefoundintheBasicTutorialfolderontheFlowJoCDorat:(FITC.)Thegoalofthistitrationexperimentistodeter-minetheconcentrationoftheanti-CD8-FITCreagentthatachievessaturatingstaining;i.e.,maximalstainingoftheCD8Tcellsunderdefinedconditions(20minutesatroomtemperature).Theconcentrationofstainingreagentsrecommendedbythemanufacturercanbesignificantlyinexcessofthesaturatingconcentrationrequiredforyourexperimen-talconditions.Atitrationexperimentlikethisonecanconserveyourreagentsandsavemoney.12Thedataanalysisinthisexperimentconsistsofidentifyingthesubpopulationofcellsthatarestainedwithanti-CD8antibodyandcalculatingamedianfluorescenceintensity(MFI)statisticonthissubpopulation.Thecomputedstatisticswillbeusedtodeterminethemosteconomicalconcentrationofanti-CD8-FITCreagenttouseinsubsequentexperiments.ExperimentalMethodHumanperipheralbloodmononuclearcellpreparationfromasinglehealthydonorwasdividedintoeighttubes.Thefirsttubeistheunstainedcontrol.Thesubsequentseventubeswerestainedwithaserialtwo-folddilutionoftheanti-CD8-FITCreagentstartingat2μg/ml.Cellswerestainedfor20minutes,washedandresuspendedinstainingmedium.Thedatafileswereacquiredfromthecytometer,with10,000eventscollectedforeachtube.ThisdatacanbefoundontheFlowJoCDinSampleDataFiles\BasicTutorialData_Workspaces\BasicTutorialData.Youcandownloaditfrom(?)buttonormenuitem,whichisfoundinallwindowsanddialogboxes.Thisbuttonlaunchesanetworkbrowserandtakesyoudirectlytothewebpagerelevanttothewindowyouareworkingon.Toviewtheentirereferencemanual,pointyourwebbrowserto::asetoffunctionbuttonsatthetop,aGroupPanel,andaSamplePanel.Whenyouselecttubesandmakeagroup,thegroupnameislistedinthegrouppanel.Whenyouclickonagroup,allthesamplesthatbelongtothatgroupcanbeseeninthesamplepanel.TheAllSamplesgroupiscreatedautomati-cal