3 panel design

23-13536-00Rev.01PanelDesignPanelDesign23-13536-00Rev.012ResearchPanelDesignExampleIntracellularCytokineStaining(ICS)ICSquantifiesantigen-specificT-cellresponsesinsettingssuchasexperimentalvaccination.•QualityandquantityofrespondingTcells•CD4+andCD8+cellsmakingmultiplecytokines•CD4+andCD8+differentiationstage(memory/effectormarkers)PanelDesign23-13536-00Rev.013ICSMarkers•Bothcytokinesandmemory/effectormarkersareimportant.•Evidencesuggeststoinclude:–CD3,CD4,CD8,IFN-g,IL-2,viabilitydye•Memorymarkerscaninclude:–CD45RA,CD27,CD28,andCCR7•Additionalmarkersofinterest:–TNF-a,CD107–CD95,CD57,CD127PanelDesign23-13536-00Rev.014ExamplePanelGoals•QuantifyCD4+andCD8+naïveandmemorysubsets•QuantifyCD4+CD25++(Tregs)•Evaluatecytokineproductionuponantigenstimulation•PaneltoberunonBDFACSCantoIIorBDLSRIIcytometerwithdefault4-2-2configurationViabilityDye–CD3+CD8+IFN-γIL-2IFN-γIL-2CD45RA+naiveCD45RA–memoryCD4+IFN-γIL-2IFN-γIL-2CD45RA+naiveCD45RA–memoryCD25++TregsPanelDesign23-13536-00Rev.015ICSPanel:FirstAttemptAresearcherchecksthefridgeandfindssomereagentsleftoverfromotherprojects.viabilitydye7-AAD(PerCP-Cy5.5)CD3APCCD4AmCyanCD8FITCCD25APC-Cy7CD45RAPE-Cy7IFN-gPEIL-2V450PanelDesign23-13536-00Rev.016ICSPanel:FirstAttemptDataMostpopulationswereidentified…CD3APCCD45RAPE-Cy7IFNgPE7-AADCD4AmCyanSSCSSCCD3APCCD8FITCCD45RAPE-Cy7PanelDesign23-13536-00Rev.017ICSPanel:FirstAttemptDataHowever...CD4AmCyanCD45RAPE-Cy7CD25APC-Cy7CD25APC-Cy7IFNgPEIL-2V450IFNgPEIL-2V450CD4+CD25+populationisnotclearlydefined(plotsaregatedonCD3+).ResearcherwouldlikebetterseparationforIL-2andIFN-g.PanelDesign23-13536-00Rev.018PanelDesignIssues•Someantigensareexpressedathighlevels,someatlowlevels,someoverarange.•Somedyesarebright,othersaredim.•Emissionspilloverfrombrightmarkerscandegradethesensitivityofdimmarkers.•Someantigenmarkersareavailableonlyincertaincolors.•Tandemdyesmaybeunstable.•Assaycomplexityincreaseslikelihoodoferror.PanelDesign23-13536-00Rev.019KnowYourCytometerMarkersensitivityandspilloverareaffectedby:•Laserwavelengthandpower.•Detectorrangeandefficiency.PanelDesign23-13536-00Rev.0110PanelDesignGoals•Obtainsufficientmeasurementsensitivityinallchannels.•Ensurethatbrightpositivepopulationscanbeaccuratelycompensatedandkeptwithinthelinearrangeofthescale.•Avoidfalsepositivesandotherartifactsoftandemdyedegradation.PanelDesign23-13536-00Rev.0111PanelDesignPrinciples1.Matchbrighterfluorochromeswithlower-expressedantigensandvice-versa.•Determinetheantigensandclassifyexpression.•Determineafluorochromesetandclassifybrightness.•Matchantigenswithfluorochromes.2.Avoidsignificantspectraloverlapbetweenmarkersonthesamecellpopulation.•Spreadthemarkersacrossasmanylasersaspossible.•Avoidsignificantspilloverofamarkerintoanotherchannelusedwiththesamecellpopulation—especiallyintoachannelwithalowersignal.3.Preferred-laserfluorochromesformarkersonhighlyautofluorescentcells.4.Checkfortandemdyeissues.PanelDesign23-13536-00Rev.0112CheckMarkerAvailabilityviabilitydyexxxxxxCD3xxxxxxxxCD4xxxxxxxxCD8xxxxxxxxCD25xxxxxxCD45RAxxxxxIFN-gxxxxIL-2xxxxxx–availableBDFACSelectMulticolorPanelDesigner•Measureofantigenexpression.•Canvaryduetocellactivationlevelandfunctionaldifferences.•Canbearange(smearedpopulation).PhenotypeAntigenMoleculesperCellx1000TcellCD255CD3124CD590CD720CD45200CD4+TcellCD4100CD2820CCR54–24CD8+TcellCD890CD2815PanelDesign23-13536-00Rev.0114IdentifyandClassifytheMarkersLowexpression,significantvariability,rareevents,oruncharacterizedantigensHighorintermediateexpression,oftenoveracontinuum(smear).Highexpression,easilyclassifiedpositiveandnegativeCD3CD4CD8CD45RAIFN-γIL-2CD25PanelDesign23-13536-00Rev.0115ClassifyFluorochromeBrightnessPEAPCPE-Cy7FITCPerCP-Cy5.5APC-Cy7APC-H7V450AmCyanBrightfluorochromesMid-levelfluorochromesDimfluorochromesPanelDesign23-13536-00Rev.0116viabilitydyeCD45RAPE-Cy7MatchAntigenswithFluorochromesAqua(viabilitydye)CD3V450CD8APCH7CD4PerCP-Cy5.5IFN-γFITCIL-2APCCD25PEAmCyanV450APC-Cy7APC-H7PerCP-Cy5.5FITCPE-Cy7APCPECD3CD4CD8CD45RAIFN-γIL-2CD25PanelDesign23-13536-00Rev.0117ICSPanel:SecondAttemptAfterreassigningfluorochromesbybrightnessandmarkerexpressiontheresearcherobtained:viabilitydyeAqua(AmCyanchannel)CD3V450CD4PerCP-Cy5.5CD8APC-H7CD25PECD45RAPE-Cy7IFN-gFITCIL-2APCPanelDesign23-13536-00Rev.0118ICSPanel:SecondAttemptDataAquaCD3V450SSCSSCCD8APC-H7FirstAttemptSecondAttemptCD4PerCP-Cy5.57-AADCD4AmCyanSSCSSCCD3APCCD8FITCPanelDesign23-13536-00Rev.0119ICSPanel:SecondAttemptDataCD4PerCP-Cy5.5CD25PEIFNgFITCIL-2APCFirstAttemptSecondAttemptCD4AmCyanCD25APC-Cy7IFNgPEIL-2V450PanelDesign23-13536-00Rev.0120AvoidSpectralOverlapWhenmultiplemarkersarepresentonacellpopulation:•Spreadthemarkersacrossasmanylasersaspossible.•Avoidsignificantspilloverofabrightmarkerintoadimmarker.PanelDesign23-13536-00Rev.0121Spilloveron4-2-2ConfigurationPanelDesign23-13536-00Rev.0122ICSPanel:SecondAttemptDataCD4PerCP-Cy5.5CD25PEwithoutPE-Cy7ResearchersuspectsthatspilloverfromCD45RAPE-Cy7ishavin