流式細(xì)胞技術(shù)熒光抗體

FluorochromesFluorochromePropertiesDesirablepropertiesforfluorochromes:HighrelativebrightnessNarrowemissionspectrum

(lowspectraloverlapincombination)Easilyconjugated(forimmunophenotyping)Fluorochromescanbecharacterizedby:TypeofmoleculeExcitationandemissionwavelengthsRelativebrightness2FluorochromeMoleculeTypesSmallorganicmolecules

examples:FITC,BDHorizonTMV450,Cy7Fluorescentproteins

examples:PE,APC,PerCPTandemdyes—typically,thecouplingofafluorescentproteindonorwithasmallorganicmoleculeacceptor

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

examples:Qdot565,Qdot6053SomeCommonFluorochromesFluorochromeTypeofmoleculeFluoresceinisothyocyanate(FITC)SmallorganicAlexaFluor?488SmallorganicPhycoerythrin(PE)ProteinPE-CyTM5ProteintandemPeridininchlorophyllprotein(PerCP)ProteinPerCP-CyTM5.5ProteintandemPE-CyTM7ProteintandemAllophycocyanin(APC)ProteinAlexaFluor?647SmallorganicAPC-CyTM7ProteintandemBDTMAPC-H7ProteintandemBDHorizonTMV450SmallorganicPacificBlueTMSmallorganicBDHorizonTMV500SmallorganicAmCyanProtein4SmallOrganicFluorochromesAdvantagesLowmolecularweightEasytoconjugate—directattachmenttofreeaminogroupsonmAbExcellentstabilityExtremelyconsistentemissionspectraDisadvantagesSmallStokesShift(50?100nm)Tendtobelessbright5ProteinFluorochromesAdvantagesGoodstabilityConsistentemissionspectraMediumStokesShift(75?200nm)TendtobemorebrightDisadvantagesHighmolecularweightMoredifficulttoconjugate—intermediariesneededtoattachtomAb6TandemDyeFluorochromesAdvantagesVerylargeStokesShift(150?300nm)Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantagesHighmolecularweight(similartofluorescentprotein)Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)Hardertoconjugate(sameasfluorescentprotein)Sometandemshavepoorstability7NanocrystalFluorochromesAdvantagesLargeStokesShift(100?500nm)TendtobeverybrightEmissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsourceHighlyresistanttophotobleachingNanocrystalssharebiophysicalandconjugationproperties

DisadvantagesDifficulttoconjugateInstabilityofbindingsCytotoxicityWideexcitationrangeproducescross-laserspillover8ExcitationandEmissionExcitationwavelengthsdeterminelasersthatcanexcitethefluorochrome.EmissionwavelengthsdeterminefiltersandPMTsthatcanmeasuretheemissionsignal.9KnowYourCytometerCytometerConfigurationBDFACSCantoTMII4-2-2configurationisshownbelowBDTMLSRII4-2-2configurationissimilar4detectors

forbluelaser2detectors

forredlaser2detectors

forvioletlaser10TypicalExcitationandEmission

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-Cy711FluorochromeUseDependson

theCytometerConfiguration6-color8-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-dots12FluorochromeBrightnessThebrightnessofafluorochromedependsontwofactors:MolarExtinctionCoefficient()measureshowwellafluorochromeabsorbsenergy.QuantumYield(Qy)istheratioofphotonsemittedtophotonsabsorbed.Brightness=

x

QyRelativeBrightness=BrightnessofPEBrightness13SomeFluorochromesareMUCHBrighterPEis~50xbrighterthanFITCand~10xbrighterthanAPC.APCis~5xbrighterthanPacificBlue?.Extinctioncoefficientismoresignificantthanquantumyieldindeterminingbrightness.FluorochromeMolarExtinctionCoefficient(mol-1xcm-1)QuantumYieldRelativeBrightness(toPE)PE19600000.98100.00%PE-Cy51960000<0.881.63%PerCP320230NA16.66%APC2320230.688.21%FITC670000.501.74%PacificBlue?360000.801.5%14DD=differencebetweenthemediansofthepositiveandnegativepopulationsW

=spread(2xrSD)ofthenegativepopulationStainIndexStainIndex=Stainindexisapracticalwaytocharacterizethebrightnessofamarkerwithrespecttoagivenopticalconfiguration.DWWStainindexcanalsobeusedtocharacterizethesensitivityofafluoresenceparameter.15TypicalCD4StainIndexes—BDLSRIIFluorochromeCD4CloneFilterSetStainIndexRelativeBrightnessPERPA-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.16TypicalCD4StainIndexes—BDFACSCantoIIFluorochromeCD4CloneFilterSetStainIndexRelativeBrightnessPESK3585/42467100.00%PE-Cy5———81.63%PerCPSK3695/405916.66%APCSK3660/203848.21%FITCSK3530/30581.74%PacificBlue?SK3450/50131.5%FITChasapproximatelythesameCD4stainindexasPerCP,butapproximately1/10ththerelativebrightness.17StainIndexFactorsStainindexisdependentontheopticalconfigurationandadditionalperformancefactors.Factorsthatcanaffectstainindexinclude:LaserwavelengthandpowerDetectorrangeDetectorefficiencyBackgroundsignalDon’tdependonpublishedvalues—measurestainindexonyourownsystem.18CD4StainIndexesAcrossCytometersStainIndexExercise19FluorescenceSpilloverEmissionofFITC

inPEchannel20SignificantSpilloverson4-2-2Configuration21SpilloverDecreasesSensitivityWithoutCD45AmCyanWithCD45AmCyanCD19FITCSpillovercansignificantlyincreasethevariabilityofnegativeanddimpopulations,evenaftercompensationisapplied.22LostPopulationduetoSpilloverLymphocytesstainedwithCD45FITCandCD4PECD45FITCcausesdimCD4+CD45+

tobedifficulttodistinguishdueto

significantFITCspilloverintoPE.LymphocytesstainedwithCD45PerCPandCD4PECD45PerCPallowsdimCD4+CD45+

tobedistinguishedfrombackground

duetominimalPerCP

spilloverintoPE.23TandemDyeIssuesUsetandemdyesinresearchapplicationswithconsiderationoftheirtechnicallimitations.APC-Cy7(andtoalesserextentPE-Cy7)candegradeinthepresenceoflight,fixation,andelevatedtemperature.DegradationcausesloweremissionintheCy7detectorandhigheremissioninthedetectoroftheparentdye(APCorPE).APC-H7,APC-Cy7,andPE-Cy7performancecanbeaffectedbypolystyrenetubes.24TandemDegradation—FalsePositivesFalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7WithCD8APC-Cy7WithoutCD8APC-Cy725TandemDegradationOverTime0hours~2hours~20hoursPECD3PE-Cy5CD8PE-Cy7TimeSample

LeftinLightPEPE26APC-H7IsMoreStableThanAPC-Cy7ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy727TandemDyeRecommendationsWhenusingAPC-Cy7andPE-Cy7,bewareoffixativeandlightinstabilityissues.IfproblemsarisewhenusingpolystyrenewithAPC-H7,APC-Cy7,orPE-Cy7,tryswitchingtoK-resinorpolycarbonate.PerCP-Cy5.5islesssusceptibletoinstabilitythanAPC-Cy7andPE-Cy7.BDoffersAPC-H7conjugatedantibodiesthataremorestablethanAPC-Cy7conjugatesandhavelessspilloverintotheAPCdetector.28NewVioletLaserFluorochromesBDHorizonV450Maximumexcitationat404nm,emissionpeakof448nm.BD

HorizonV450reagentsexhibitperformancecomparabletoPacificBlue?reagentsasmeasuredbyStainIndex.ImprovedstabilitywithfixationcomparedtoPacificBlue?.29NewVioletLaserFluorochromesBDHorizonV500Maximumexcitationat415nm,emissionpeakof500nm.ProvidessignificantlyreducedspilloverintotheFITCchannelcomparedtoAmCyan—V500haslowexcitationwiththebluelaser.ImprovedstabilitywithfixationcomparedtoAmCyan.30BDHorizonV500LowSpilloverintoFITCV500stainedcellsAmCyanstainedcellsDataisuncompensated.31FactorsAffectingReagentPerformanceRelativebrightnessofthefluorochromeNumberoffluorochromesperantibodyExpressionlevelson(orin)thecellsofinterestBackgroundcontributionsSpilloverPhotostabilityReagentenvironmentCytometerconfiguration32Non-ConjugatedFluorescentDyesThesedyesbinddirectlytocellcomponents.Viabilitydyesdiscriminatelivece