流式细胞(FACS)多色分析

MulticolorAnalysisWearenowgoingtotalkabouttoolsandstrategiesusefulwhenitcomestodataanalysis.AnalysisMulticolorAnalysisStrategies Plottypes Hierarchicalgating Populationhierarchies Backgating1stbullet;Everyanalysissoftwarewillgiveyouanoptiontolookatthedatausingdifferentplottypes.Peoplehavetheirownpreferencesbutwewilldiscusstheadvantagesofsomeplottypesoverothers.2ndbullet:it’sveryimportantinmulticolorflowdataanalysis,tokeepaneyeonparentpopulationsandmakesurethegatescomesfromthedesiredparent.Lastbullet:Divaallowsyouveryeasybackgatingbyassigningdifferentcolorstoeachpopulation.Wewillseeanexample.AnalysisUseDifferentPlotTypesHistograms—usinghistogramscanhelpdiscriminatebetweentwopopulations.DotPlot:hardtodiscriminatepopulationsDotPlot:displayingfewereventsimprovesdiscriminationHistogram:populationboundaryisevidentSometimes,ithelpstoswitchplottypestolookatdataanddecidewhichworksbestforthatdataset.AnalysisUseDifferentPlotTypesContourPlots—usingcontourplotscan:Helplocatepopulationmeans.Indicateaneedforbiexponentialscaling.DotPlot:hardtoseepopulationsbunchedonaxisContourPlot:showsthatbiexponentialdisplaymightbeneededBiexponentialContourPlot:givesbestdisplayofpopulationsReadthesentence.Pointtothedotplotonleftandhownegativepopulationsappearandthenshowhowthosepopulationsareeasilyobservedonacontourplotwithoutbiexdisplay.AnalysisHierarchicalGatingInthisexampleweshowhierarchicalgating.WalkthroughthegatesstartingfromCD3+….CD4+…cytokine-positiveetc.AnalysisHierarchicalGatingWhatwentwronghere?CorrectHierarchyPopulationhierarchyavailableinDivaallowstoquicklymakesurethegatesweredrawnfromthedesiredparentpopulation.Thengothroughtheexamples.AnalysisBackGating ResearcherwantstodiscriminateTregsbasedonCD25andCD127expression. NeedtoensureagateincludestheentireFOXP3+population.Backgatinghasbeenstrategycommonlyusedforanalysis.Theideaistotrackasubset.Downtoit’sparentgatesinordertodetermineifthosegatesareproperlyset.Walkthroughtheexample.AnalysisAdditionalAnalysisStrategies Biexponentialscaling Eventstodisplay Hingedandoffsetquadrantgates Derived(boolean)gates Snap-togates Statistics AnalysistemplatesWealreadytalkedabouttheimportanceofbiexponentialscalingtovisualizenegativepopulationsontheplot.Divaallowsyoutoeditthenumberofeventstodisplayontheplot,thishelpswhiledrawingregionsspeciallyforthedensepopulations,orwhentheresolutionofpositive/negativeisnottooobvious.Indiva,wehaveanoptiontodrawhinged/offsetquadrantmarkersthathelpsforaccurateanalysiswhenthepopulationsarenotperfectlyaligned.Derivedgates:AND,ORgatesallowstocombinepopulationsSnap-togatesareusefulforautomatedanalysiswheretheregionsaredrawnbasedonthedensityofthepopulationsThereareplentyofstatisticalparametersthatcanberetrievedfromeachanalysis.Youcaneditthepopulationsorthestatisticsforeachofthepopulationsyouselect.Onceyouhavethebestgatingstrategyandstatisticsandpopulationhierarchysetyoucansavethisasyouranalysistemplateandusetoanalyzedataforseveralfilesfromthatexperiment.AnalysisICSDataAnalysisResultsInformationthatcanberetrieved:Numberofcollectedevents,numberofliveCD3+cells%ofmainpopulations:%CD3+,CD4+,CD8+%ofnaïve(CD45RA+)andmemory(CD45RA–)CD4+andCD8+cells%CD25+CD4+cells(Tregs)Forthecytokines:%ofCD4+andCD8+making1or2cytokinesAnalysisExerciseNowwearegoingtoanalyzethedatathatyouacquired.ReadtheslideTheideaisthatyouusedifferentanalysistoolswediscussedtogettheseresults.AnalysisICSPanelAnalysisHere’sanexampleofgatingstrategybuttrytobecreativeandcomeupwithdifferenttemplateusingdifferentregionshapesandgatingstrategyetc.Wearenowgoingtotalkabouttoolsandstrategiesusefulwhenitcomestodataanalysis.1stbullet;Everyanalysissoftwarewillgiveyouanoptiontolookatthedatausingdifferentplottypes.Peoplehavetheirownpreferencesbutwewilldiscusstheadvantagesofsomeplottypesoverothers.2ndbullet:it’sveryimportantinmulticolorflowdataanalysis,tokeepaneyeonparentpopulationsandmakesurethegatescomesfromthedesiredparent.Lastbullet:Divaallowsyouveryeasybackgatingbyassigningdifferentcolorstoeachpopulation.Wewillseeanexample.Sometimes,ithelpstoswitchplottypestolookatdataanddecidewhichworksbestforthatdataset.Readthesentence.Pointtothedotplotonleftandhownegativepopulationsappearandthenshowhowthosepopulationsareeasilyobservedonacontourplotwithoutbiexdisplay.Inthisexampleweshowhierarchicalgating.WalkthroughthegatesstartingfromCD3+….CD4+…cytokine-positiveetc.PopulationhierarchyavailableinDivaallowstoquicklymakesurethegatesweredrawnfromthedesiredparentpopulation.Thengothroughtheexamples.Backgatinghasbeenstrategycommonlyusedforanalysis.Theideaistotrackasubset.Downtoit’sparentgatesinordertodetermineifthosegatesareproperlyset.Walkthroughtheexample.Wealreadytalkedabouttheimportanceofbiexponentialscalingtovisualizenegativepopulationsontheplot.Divaallowsyoutoeditthenumberofeventstodisplayontheplot,thishelpswhiledrawingregionsspeciallyforthedensepopulations,orwhentheresolutionofpositive/negativeisnottooobvious.Indiva,wehaveanoptiontodrawhinged/offsetquadrantmarkersthathelpsforaccurateanalysiswhenthepopulationsarenotperfectlyaligned.Derivedgates:AND,ORgatesallowstocombinepopulationsSnap-togatesareusefulforautomatedanalysiswheretheregionsaredrawnbasedonthedensityofthepopulationsThereareplentyofstatisticalparametersthatcanberetrievedfromeachanalysis.Youcaneditthepopulationsorthestatisticsforeachofthepopulationsyouselect.Onceyouhavethebestgatingstrategyandstatisticsandpopulationhierarchysetyoucansavethisasyouranalysistemplateandusetoanalyzedataforseveralfilesfromthatexperiment.Nowwearegoingtoanalyzethedatathatyouacquired.ReadtheslideTheideaisthatyouusedifferentanalysistoolswediscussedtogettheseresults.Here’sanexampleofgatingstrategybuttrytobecreativeandcomeupwithdifferenttemplateusingdifferentregionshapesandgatingstrategyetc.