多色流式细胞(FACS)调补偿与圈门.ppt

23-13536-00Rev.01,Compensation,Controls,andDataCollection,23-13536-00Rev.01,2,CompensationGoal,RemovespilloversignalssothatsubpopulationMFIsagree,23-13536-00Rev.01,3,DoesCompensationIncreaseDataSpread?,Uncompensated,Compensated,CompensationcorrectstheMFI,butcannotremoveallofthevariationincreaseintroducedbyspillover.However,undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied.Whenviewingdataonlogandbiexponentialplots,dataspreadappearstoincreasewhencompensationisappliedthisisoftenavisualartifactduetothenon-linearscalingoftheplot.,Spread,23-13536-00Rev.01,4,CytometerSettingsWorkflow,ObtainCS&Tsettings,Applyapplicationsettings,Verifysettingswithsample,Calculatecompensation,23-13536-00Rev.01,5,CompensationforTandemDyes,Compensationfortandemdyeconjugatescanvary,evenbetweentwoexperimentswiththesameantibody.Tandemdyesrequirecompensationthatis:lot-specificexperiment-specificlabel-specific,23-13536-00Rev.01,6,CompensationRulesPart1,Thefluorescenceemissionspectrumofcompensationcontrolsmustmatchtheexperimentreagentsthisisespeciallycriticalwithtandemreagents.Compensationcontrolnegativeandpositivepopulationsmustbefromthesamecellorparticletype.Example:dontuseaCD3monocytenegativepopulationwithaCD3+lymphocytepositivepopulation.,23-13536-00Rev.01,7,CompensationRulesPart2,Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations.Compensationcontrolsmustplacethepositivepopulationinthelinearrange.Whenusingcellsforcompensationcontrols,increasethenumberofeventstoatleast10,000pertube.WheneverMFItargetvalueschange,reruncompensation.,23-13536-00Rev.01,8,BDCompBeads,Usethesameantibodiesasintheexperimentalsamples.Createbrightanduniformpositivefluorescencepeaks.Avoidusinglimitedsample.Beadsarecoatedwithanti-mousekappa*.*BDCompBeadsarealsoavailablecoatedwithanti-ratkappaandanti-hamsterkappa.,CompBead,Aconvenientwaytocreateaccuratesingle-colorcompensationcontrols,23-13536-00Rev.01,9,UnstainedCompensationControlTube,WhenshouldIuseanunstainedcompensationcontroltube?InBDFACSDivasoftware:Ifusingaseparateunstainedcontrol,selecttheIncludeseparateunstainedcontroltube/wellcheckbox.Ifnotusingaseparateunstainedcontrol,clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation.,23-13536-00Rev.01,10,CompensationQC,Biexponentialdisplayrevealscompensationproblems.,Over,Correct,Biexponential,23-13536-00Rev.01,11,CompensationDiscussionPoints,Howoften?HowtoQCandadjustsettings?Postacquisition?Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples?(example:fixedandpermeabilized),23-13536-00Rev.01,12,Controls,23-13536-00Rev.01,13,ChooseAppropriateControls,23-13536-00Rev.01,14,GatingControls,Fluorescence-Minus-One(FMO)controlIncludesalltestantibodiesexcepttheoneofinterest.Doesnttakebackgroundstainingintoaccount.Usefulinsettinggatesandconfirmingspilloverproblems.IsotypecontrolNon-specificantibodyofsameisotypeasthetestantibody.Doesnttakespilloverintoaccount.CombinedcontrolAlltestantibodiesexcepttheoneofinterest,whichisreplacedbyanisotypecontrol.Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody.,23-13536-00Rev.01,15,FMOExample,FMOAmCyan,23-13536-00Rev.01,16,ComparisonofGatingControls,23-13536-00Rev.01,17,DataCollection,23-13536-00Rev.01,18,125,000lymphocytescollected,20,000lymphocytescollected,NumberofEventsvsMeasurementPrecision,CD4+Tcells,CD8+Tcells,14events=0.14%,73events=0.34%,8events=0.23%,51events=0.09%,23-13536-00Rev.01,19,StatisticalSignificanceofResults,DeterminingtheNumberofEventstoCollect,NumberofRelevantEventstoCollect,%Background(False+),Lowest%Positive,90%power,p0.05,99%power,p0.005,0.01,0.02,260,000,720,000,0.01,0.05,32,000,90,000,0.01,12,000,32,000,0.02,0.05,67,000,190,000,0.02,16,000,45,000,0.03,0.05,170,000,480,000,0.03,0.1,23,000,63,000,0.04,0.1,33,000,93,000,0.05,0.1,52,000,140,000,0.06,0.1,86,000,240,000,0.07,0.1,160,000,450,000,0.08,0.2,17,000,46,000,0.1,0.2,26,000,72,000,0.1,0.1,23-13536-00Rev.01,20,DataCollectionDiscussion,StorageGatesandStoppingGatesGlobalWorksheetsvsNormalWorksheetsTemplates,23-13536-00Rev.01,21,Experim