流式细胞技术荧光抗体

流式细胞技术荧光抗体23-13536-00Rev.01第1页，课件共35页，创作于2023年2月FluorochromePropertiesDesirablepropertiesforfluorochromes:HighrelativebrightnessNarrowemissionspectrum

(lowspectraloverlapincombination)Easilyconjugated(forimmunophenotyping)Fluorochromescanbecharacterizedby:TypeofmoleculeExcitationandemissionwavelengthsRelativebrightness223-13536-00Rev.01第2页，课件共35页，创作于2023年2月FluorochromeMoleculeTypesSmallorganicmolecules

examples:FITC,BDHorizonTMV450,Cy7Fluorescentproteins

examples:PE,APC,PerCPTandemdyes—typically,thecouplingofafluorescentproteindonorwithasmallorganicmoleculeacceptor

examples:PE-Cy7,PerCP-CyTM5.5Nanocrystals(Qdots)—inorganicsemiconductors

examples:Qdot565,Qdot605323-13536-00Rev.01第3页，课件共35页，创作于2023年2月SomeCommonFluorochromesFluorochromeTypeofmoleculeFluoresceinisothyocyanate(FITC)SmallorganicAlexaFluor®488SmallorganicPhycoerythrin(PE)ProteinPE-CyTM5ProteintandemPeridininchlorophyllprotein(PerCP)ProteinPerCP-CyTM5.5ProteintandemPE-CyTM7ProteintandemAllophycocyanin(APC)ProteinAlexaFluor®647SmallorganicAPC-CyTM7ProteintandemBDTMAPC-H7ProteintandemBDHorizonTMV450SmallorganicPacificBlueTMSmallorganicBDHorizonTMV500SmallorganicAmCyanProtein423-13536-00Rev.01第4页，课件共35页，创作于2023年2月SmallOrganicFluorochromesAdvantagesLowmolecularweightEasytoconjugate—directattachmenttofreeaminogroupsonmAbExcellentstabilityExtremelyconsistentemissionspectraDisadvantagesSmallStokesShift(50−100nm)Tendtobelessbright523-13536-00Rev.01第5页，课件共35页，创作于2023年2月ProteinFluorochromesAdvantagesGoodstabilityConsistentemissionspectraMediumStokesShift(75−200nm)TendtobemorebrightDisadvantagesHighmolecularweightMoredifficulttoconjugate—intermediariesneededtoattachtomAb623-13536-00Rev.01第6页，课件共35页，创作于2023年2月TandemDyeFluorochromesAdvantagesVerylargeStokesShift(150−300nm)Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantagesHighmolecularweight(similartofluorescentprotein)Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)Hardertoconjugate(sameasfluorescentprotein)Sometandemshavepoorstability723-13536-00Rev.01第7页，课件共35页，创作于2023年2月NanocrystalFluorochromesAdvantagesLargeStokesShift(100−500nm)TendtobeverybrightEmissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsourceHighlyresistanttophotobleachingNanocrystalssharebiophysicalandconjugationproperties

DisadvantagesDifficulttoconjugateInstabilityofbindingsCytotoxicityWideexcitationrangeproducescross-laserspillover823-13536-00Rev.01第8页，课件共35页，创作于2023年2月ExcitationandEmissionExcitationwavelengthsdeterminelasersthatcanexcitethefluorochrome.EmissionwavelengthsdeterminefiltersandPMTsthatcanmeasuretheemissionsignal.923-13536-00Rev.01第9页，课件共35页，创作于2023年2月KnowYourCytometerCytometerConfigurationBDFACSCantoTMII4-2-2configurationisshownbelowBDTMLSRII4-2-2configurationissimilar4detectors

forbluelaser2detectors

forredlaser2detectors

forvioletlaser1023-13536-00Rev.01第10页，课件共35页，创作于2023年2月TypicalExcitationandEmission

BDFACSCantoII4-2-2(BDLSRII4-2-2issimilar)Detector

RangeVioletLaser

405nmBlueLaser

488nmRedLaser

633nm410–490nmBDHorizonV450

PacificBlueTM500–560nmBDHorizonV500

AmCyan515–545nmFITC

AlexaFluor®488564–606nmPE650–670nmAPC

AlexaFluor®647670–735nmPerCP-Cy5.5

PE-Cy5

PerCP750–810nmPE-Cy7BDAPC-H7

APC-Cy71123-13536-00Rev.01第11页，课件共35页，创作于2023年2月FluorochromeUseDependson

theCytometerConfiguration

6-color

8-color

Morethan

8colorsFITC,AlexaFluor®488FITC,AlexaFluor®488FITC,AlexaFluor®488PEPEPEPE-TexasRed®,PE-AlexaFluor®610,orPE-AlexaFluor®594PE-Cy5,PerCP,

orPerCP-Cy5.5PE-Cy5,PerCP,

orPerCP-Cy5.5PE-Cy5,PerCP,

orPerCP-Cy5.5PE-Cy7PE-Cy7PE-Cy7APC,AlexaFluor®647APC,AlexaFluor®647APC,AlexaFluor®647APC-Cy5.5,AlexaFluor®680,

AlexaFluor®700APC-H7,APC-Cy7APC-H7,APC-Cy7APC-H7,APC-Cy7BDHorizonV500,AmCyanBDHorizonV500,AmCyanBDHorizonV450,PacificBlue™BDHorizonV450,PacificBlue™PacificOrange™,Q-dots1223-13536-00Rev.01第12页，课件共35页，创作于2023年2月FluorochromeBrightnessThebrightnessofafluorochromedependsontwofactors:MolarExtinctionCoefficient(

)measureshowwellafluorochromeabsorbsenergy.QuantumYield(Qy)istheratioofphotonsemittedtophotonsabsorbed.Brightness=

x

QyRelativeBrightness=BrightnessofPEBrightness1323-13536-00Rev.01第13页，课件共35页，创作于2023年2月SomeFluorochromesareMUCHBrighterPEis~50xbrighterthanFITCand~10xbrighterthanAPC.APCis~5xbrighterthanPacificBlue™.Extinctioncoefficientismoresignificantthanquantumyieldindeterminingbrightness.FluorochromeMolarExtinctionCoefficient(mol-1xcm-1)QuantumYieldRelativeBrightness(toPE)PE19600000.98100.00%PE-Cy51960000<0.881.63%PerCP320000NA16.66%APC2320000.688.21%FITC670000.501.74%PacificBlue™360000.801.5%1423-13536-00Rev.01第14页，课件共35页，创作于2023年2月DD=differencebetweenthemediansofthepositiveandnegativepopulationsW

=spread(2xrSD)ofthenegativepopulationStainIndexStainIndex=Stainindexisapracticalwaytocharacterizethebrightnessofamarkerwithrespecttoagivenopticalconfiguration.DWWStainindexcanalsobeusedtocharacterizethesensitivityofafluoresenceparameter.1523-13536-00Rev.01第15页，课件共35页，创作于2023年2月TypicalCD4StainIndexes—BDLSRII

FluorochromeCD4CloneFilterSetStainIndexRelativeBrightnessPERPA-T4585/40305100.00%PE-Cy5RPA-T4695/4019881.63%PerCPRPA-T4695/403016.66%APCRPA-T4660/202638.21%FITCRPA-T4530/30431.74%PacificBlue™RPA-T4440/40631.5%APChasahigherCD4stainindexthanPE-Cy5,

butapproximately1/10ththerelativebrightness.1623-13536-00Rev.01第16页，课件共35页，创作于2023年2月TypicalCD4StainIndexes—BDFACSCantoII

FluorochromeCD4CloneFilterSetStainIndexRelativeBrightnessPESK3585/42467100.00%PE-Cy5———81.63%PerCPSK3695/405916.66%APCSK3660/203848.21%FITCSK3530/30581.74%PacificBlue™SK3450/50131.5%FITChasapproximatelythesameCD4stainindexasPerCP,butapproximately1/10ththerelativebrightness.1723-13536-00Rev.01第17页，课件共35页，创作于2023年2月StainIndexFactorsStainindexisdependentontheopticalconfigurationandadditionalperformancefactors.Factorsthatcanaffectstainindexinclude:LaserwavelengthandpowerDetectorrangeDetectorefficiencyBackgroundsignalDon’tdependonpublishedvalues—measurestainindexonyourownsystem.1823-13536-00Rev.01第18页，课件共35页，创作于2023年2月CD4StainIndexesAcrossCytometersStainIndexExercise1923-13536-00Rev.01第19页，课件共35页，创作于2023年2月FluorescenceSpilloverEmissionofFITC

inPEchannel2023-13536-00Rev.01第20页，课件共35页，创作于2023年2月SignificantSpilloverson4-2-2Configuration2123-13536-00Rev.01第21页，课件共35页，创作于2023年2月SpilloverDecreasesSensitivityWithoutCD45AmCyanWithCD45AmCyanCD19FITCSpillovercansignificantlyincreasethevariabilityofnegativeanddimpopulations,evenaftercompensationisapplied.2223-13536-00Rev.01第22页，课件共35页，创作于2023年2月LostPopulationduetoSpilloverLymphocytesstainedwithCD45FITCandCD4PECD45FITCcausesdimCD4+CD45+

tobedifficulttodistinguishdueto

significantFITCspilloverintoPE.LymphocytesstainedwithCD45PerCPandCD4PECD45PerCPallowsdimCD4+CD45+

tobedistinguishedfrombackground

duetominimalPerCP

spilloverintoPE.2323-13536-00Rev.01第23页，课件共35页，创作于2023年2月TandemDyeIssuesUsetandemdyesinresearchapplicationswithconsiderationoftheirtechnicallimitations.APC-Cy7(andtoalesserextentPE-Cy7)candegradeinthepresenceoflight,fixation,andelevatedtemperature.DegradationcausesloweremissionintheCy7detectorandhigheremissioninthedetectoroftheparentdye(APCorPE).APC-H7,APC-Cy7,andPE-Cy7performancecanbeaffectedbypolystyrenetubes.2423-13536-00Rev.01第24页，课件共35页，创作于2023年2月TandemDegradation—FalsePositivesFalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7WithCD8APC-Cy7WithoutCD8APC-Cy72523-13536-00Rev.01第25页，课件共35页，创作于2023年2月TandemDegradationOverTime0hours~2hours~20hoursPECD3PE-Cy5CD8PE-Cy7TimeSample

LeftinLightPEPE2623-13536-00Rev.01第26页，课件共35页，创作于2023年2月APC-H7IsMoreStableThanAPC-Cy7ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy72723-13536-00Rev.01第27页，课件共35页，创作于2023年2月TandemDyeRecommendationsWhenusingAPC-Cy7andPE-Cy7,bewareoffixativeandlightinstabilityissues.IfproblemsarisewhenusingpolystyrenewithAPC-H7,APC-Cy7,orPE-Cy7,tryswitchingtoK-resinorpolycarbonate.PerCP-Cy5.5islesssusceptibletoinstabilitythanAPC-Cy7andPE-Cy7.BDoffersAPC-H7conjugatedantibodiesthataremorestablethanAPC-Cy7conjugatesandhavelessspilloverintotheAPCdetector.2823-13536-00Rev.01第28页，课件共35页，创作于2023年2月NewVioletLaserFluorochromesBDHorizonV450Maximumexcitationat404nm,emissionpeakof448nm.BD

HorizonV450reagentsexhibitperformancecomparabletoPacificBlue™reagentsasmeasuredbyStainIndex.ImprovedstabilitywithfixationcomparedtoPacificBlue™.2923-13536-00Rev.01第29页，课件共35页，创作于2023年2月NewVioletLaserFluorochromesBDHorizonV500Maximumexcitationat415nm,emissionpeakof500nm.ProvidessignificantlyreducedspilloverintotheFITCchannelcomparedtoAmCyan—V500haslowexcitationwiththebluelaser.ImprovedstabilitywithfixationcomparedtoAmCyan.3023-13536-00Rev.01第30页，课件共35页，创作于2023年2月BDHorizonV500LowSpilloverintoFITCV500stainedcellsAmCyanstainedcellsDataisuncompensated.3123-13536-00Rev.01第31页，课件共35页，创作于2023年2月FactorsAffectingReagentPerformanceRelativebrightnessofthefluorochromeNumberoffluorochromesperantibodyExpressionlevelson(orin)thecellsofinterestBackgroundcontributionsSpilloverPhotostabilityReagentenvironmentCytometerconfiguration3223-13536-00Rev.01第32页，课件共35页，创作于2023年2月Non-ConjugatedFluorescentDyesThesedyesbinddirectlytocellcomponents.Viabi