多色流式细胞(FACS)调补偿与圈门.ppt

Compensation Controls andDataCollection 2 CompensationGoal RemovespilloversignalssothatsubpopulationMFIsagree 3 DoesCompensationIncreaseDataSpread Uncompensated Compensated CompensationcorrectstheMFI butcannotremoveallofthevariationincreaseintroducedbyspillover However undernormalconditionsthespreadinafluorescencemeasurementwithspilloverwilldecreasewhencompensationisapplied Whenviewingdataonlogandbiexponentialplots dataspreadappearstoincreasewhencompensationisapplied thisisoftenavisualartifactduetothenon linearscalingoftheplot Spread 4 CytometerSettingsWorkflow ObtainCS Tsettings Applyapplicationsettings Verifysettingswithsample Calculatecompensation 5 CompensationforTandemDyes Compensationfortandemdyeconjugatescanvary evenbetweentwoexperimentswiththesameantibody Tandemdyesrequirecompensationthatis lot specificexperiment specificlabel specific 6 CompensationRules Part1 Thefluorescenceemissionspectrumofcompensationcontrolsmustmatchtheexperimentreagents thisisespeciallycriticalwithtandemreagents Compensationcontrolnegativeandpositivepopulationsmustbefromthesamecellorparticletype Example don tuseaCD3 monocytenegativepopulationwithaCD3 lymphocytepositivepopulation 7 CompensationRules Part2 Compensationcontrolsmustbebrightenoughtoobtaingoodseparationbetweenthepositiveandnegativepopulations Compensationcontrolsmustplacethepositivepopulationinthelinearrange Whenusingcellsforcompensationcontrols increasethenumberofeventstoatleast10 000pertube WheneverMFItargetvalueschange reruncompensation 8 BDCompBeads Usethesameantibodiesasintheexperimentalsamples Createbrightanduniformpositivefluorescencepeaks Avoidusinglimitedsample Beadsarecoatedwithanti mousekappa BDCompBeadsarealsoavailablecoatedwithanti ratkappaandanti hamsterkappa CompBead Aconvenientwaytocreateaccuratesingle colorcompensationcontrols 9 UnstainedCompensationControlTube WhenshouldIuseanunstainedcompensationcontroltube InBDFACSDivasoftware Ifusingaseparateunstainedcontrol selecttheIncludeseparateunstainedcontroltube wellcheckbox Ifnotusingaseparateunstainedcontrol clearthecheckboxandforeachparameterincludeaP3gateforthenegativepopulation 10 CompensationQC Biexponentialdisplayrevealscompensationproblems Over Correct Biexponential 11 CompensationDiscussionPoints Howoften HowtoQCandadjustsettings Postacquisition Shouldcompensationcontrolsbetreatedthesameasexperimentalsamples example fixedandpermeabilized 12 Controls 13 ChooseAppropriateControls 14 GatingControls Fluorescence Minus One FMO controlIncludesalltestantibodiesexcepttheoneofinterest Doesn ttakebackgroundstainingintoaccount Usefulinsettinggatesandconfirmingspilloverproblems IsotypecontrolNon specificantibodyofsameisotypeasthetestantibody Doesn ttakespilloverintoaccount CombinedcontrolAlltestantibodiesexcepttheoneofinterest whichisreplacedbyanisotypecontrol Mightnotaccuratelyrepresentthebackgroundstainingofthetestantibody 15 FMOExample FMOAmCyan 16 ComparisonofGatingControls 17 DataCollection 18 125 000lymphocytescollected 20 000lymphocytescollected NumberofEventsvsMeasurementPrecision CD4 Tcells CD8 Tcells 14events 0 14 73events 0 34 8events 0 23 51events 0 09 19 StatisticalSignificanceofResults DeterminingtheNumberofEventstoCollect NumberofRelevantEventstoCollect Background False Lowest Positive 90 power p 0 05 99 power p 0 005 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 20 DataCollectionDiscussion StorageGatesandStoppingGatesGlobalWorksheetsvsNormalWorksheetsTemplates 21 ExperimentSetupandRecord