流式細(xì)胞技術(shù)熒光抗體

流式細(xì)胞技術(shù)熒光抗體23-13536-00Rev.01第1頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月FluorochromePropertiesDesirablepropertiesforfluorochromes:HighrelativebrightnessNarrowemissionspectrum

(lowspectraloverlapincombination)Easilyconjugated(forimmunophenotyping)Fluorochromescanbecharacterizedby:TypeofmoleculeExcitationandemissionwavelengthsRelativebrightness223-13536-00Rev.01第2頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月SmallOrganicFluorochromesAdvantagesLowmolecularweightEasytoconjugate—directattachmenttofreeaminogroupsonmAbExcellentstabilityExtremelyconsistentemissionspectraDisadvantagesSmallStokesShift(50?100nm)Tendtobelessbright523-13536-00Rev.01第5頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月ProteinFluorochromesAdvantagesGoodstabilityConsistentemissionspectraMediumStokesShift(75?200nm)TendtobemorebrightDisadvantagesHighmolecularweightMoredifficulttoconjugate—intermediariesneededtoattachtomAb623-13536-00Rev.01第6頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月TandemDyeFluorochromesAdvantagesVerylargeStokesShift(150?300nm)Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantagesHighmolecularweight(similartofluorescentprotein)Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)Hardertoconjugate(sameasfluorescentprotein)Sometandemshavepoorstability723-13536-00Rev.01第7頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月NanocrystalFluorochromesAdvantagesLargeStokesShift(100?500nm)TendtobeverybrightEmissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsourceHighlyresistanttophotobleachingNanocrystalssharebiophysicalandconjugationproperties

DisadvantagesDifficulttoconjugateInstabilityofbindingsCytotoxicityWideexcitationrangeproducescross-laserspillover823-13536-00Rev.01第8頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月ExcitationandEmissionExcitationwavelengthsdeterminelasersthatcanexcitethefluorochrome.EmissionwavelengthsdeterminefiltersandPMTsthatcanmeasuretheemissionsignal.923-13536-00Rev.01第9頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月KnowYourCytometerCytometerConfigurationBDFACSCantoTMII4-2-2configurationisshownbelowBDTMLSRII4-2-2configurationissimilar4detectors

forbluelaser2detectors

forredlaser2detectors

forvioletlaser1023-13536-00Rev.01第10頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月FluorochromeBrightnessThebrightnessofafluorochromedependsontwofactors:MolarExtinctionCoefficient(

)measureshowwellafluorochromeabsorbsenergy.QuantumYield(Qy)istheratioofphotonsemittedtophotonsabsorbed.Brightness=

x

QyRelativeBrightness=BrightnessofPEBrightness1323-13536-00Rev.01第13頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月DD=differencebetweenthemediansofthepositiveandnegativepopulationsW

=spread(2xrSD)ofthenegativepopulationStainIndexStainIndex=Stainindexisapracticalwaytocharacterizethebrightnessofamarkerwithrespecttoagivenopticalconfiguration.DWWStainindexcanalsobeusedtocharacterizethesensitivityofafluoresenceparameter.1523-13536-00Rev.01第15頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月StainIndexFactorsStainindexisdependentontheopticalconfigurationandadditionalperformancefactors.Factorsthatcanaffectstainindexinclude:LaserwavelengthandpowerDetectorrangeDetectorefficiencyBackgroundsignalDon’tdependonpublishedvalues—measurestainindexonyourownsystem.1823-13536-00Rev.01第18頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月CD4StainIndexesAcrossCytometersStainIndexExercise1923-13536-00Rev.01第19頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月FluorescenceSpilloverEmissionofFITC

inPEchannel2023-13536-00Rev.01第20頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月SignificantSpilloverson4-2-2Configuration2123-13536-00Rev.01第21頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月SpilloverDecreasesSensitivityWithoutCD45AmCyanWithCD45AmCyanCD19FITCSpillovercansignificantlyincreasethevariabilityofnegativeanddimpopulations,evenaftercompensationisapplied.2223-13536-00Rev.01第22頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月LostPopulationduetoSpilloverLymphocytesstainedwithCD45FITCandCD4PECD45FITCcausesdimCD4+CD45+

tobedifficulttodistinguishdueto

significantFITCspilloverintoPE.LymphocytesstainedwithCD45PerCPandCD4PECD45PerCPallowsdimCD4+CD45+

tobedistinguishedfrombackground

duetominimalPerCP

spilloverintoPE.2323-13536-00Rev.01第23頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月TandemDegradation—FalsePositivesFalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7WithCD8APC-Cy7WithoutCD8APC-Cy72523-13536-00Rev.01第25頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月TandemDegradationOverTime0hours~2hours~20hoursPECD3PE-Cy5CD8PE-Cy7TimeSample

LeftinLightPEPE2623-13536-00Rev.01第26頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月APC-H7IsMoreStableThanAPC-Cy7ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy72723-13536-00Rev.01第27頁(yè)，課件共35頁(yè)，創(chuàng)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頁(yè)，課件共35頁(yè)，創(chuàng)作于2023年2月NewVioletLaserFluorochromesBDHorizonV450Maximumexcitationat404nm,emissionpeakof448nm.BD

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