齊魯醫(yī)學流式細胞技術熒光抗體

FluorochromePropertiesDesirablepropertiesforfluorochromes:HighrelativebrightnessNarrowemissionspectrum

(lowspectraloverlapinbination)Easilyconjugated(forimmunophenotyping)Fluorochromescanbecharacterizedby:TypeofmoleculeExcitationandemissionwavelengthsRelativebrightness123-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第1頁!FluorochromeMoleculeTypesSmallorganicmolecules

examples:FITC,BDHorizonTMV450,Cy7Fluorescentproteins

examples:PE,APC,PerCPTandemdyes—typically,thecouplingofafluorescentproteindonorwithasmallorganicmoleculeacceptor

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

examples:Qdot565,Qdot605223-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第2頁!SomeCommonFluorochromesFluorochromeTypeofmoleculeFluoresceinisothyocyanate(FITC)SmallorganicAlexaFluor?488SmallorganicPhycoerythrin(PE)ProteinPE-CyTM5ProteintandemPeridininchlorophyllprotein(PerCP)ProteinPerCP-CyTM5.5ProteintandemPE-CyTM7ProteintandemAllophycocyanin(APC)ProteinAlexaFluor?647SmallorganicAPC-CyTM7ProteintandemBDTMAPC-H7ProteintandemBDHorizonTMV450SmallorganicPacificBlueTMSmallorganicBDHorizonTMV500SmallorganicAmCyanProtein323-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第3頁!ProteinFluorochromesAdvantagesGoodstabilityConsistentemissionspectraMediumStokesShift(75?200nm)TendtobemorebrightDisadvantagesHighmolecularweightMoredifficulttoconjugate—intermediariesneededtoattachtomAb423-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第4頁!NanocrystalFluorochromesAdvantagesLargeStokesShift(100?500nm)TendtobeverybrightEmissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsourceHighlyresistanttophotobleachingNanocrystalssharebiophysicalandconjugationproperties

DisadvantagesDifficulttoconjugateInstabilityofbindingsCytotoxicityWideexcitationrangeproducescross-laserspillover523-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第5頁!KnowYourCytometerCytometerConfigurationBDFACSCantoTMII4-2-2configurationisshownbelowBDTMLSRII4-2-2configurationissimilar4detectors

forbluelaser2detectors

forredlaser2detectors

forvioletlaser623-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第6頁!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-dots723-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第7頁!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%823-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第8頁!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.923-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第9頁!StainIndexFactorsStainindexisdependentontheopticalconfigurationandadditionalperformancefactors.Factorsthatcanaffectstainindexinclude:LaserwavelengthandpowerDetectorrangeDetectorefficiencyBackgroundsignalDon’tdependonpublishedvalues—measurestainindexonyourownsystem.1023-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第10頁!FluorescenceSpilloverEmissionofFITC

inPEchannel1123-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第11頁!SpilloverDecreasesSensitivityWithoutCD45AmCyanWithCD45AmCyanCD19FITCSpillovercansignificantlyincreasethevariabilityofnegativeanddimpopulations,evenafterpensationisapplied.1223-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第12頁!TandemDyeIssuesUsetandemdyesinresearchapplicationswithconsiderationoftheirtechnicallimitations.APC-Cy7(andtoalesserextentPE-Cy7)candegradeinthepresenceoflight,fixation,andelevatedtemperature.DegradationcausesloweremissionintheCy7detectorandhigheremissioninthedetectoroftheparentdye(APCorPE).APC-H7,APC-Cy7,andPE-Cy7performancecanbeaffectedbypolystyrenetubes.1323-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第13頁!TandemDegradationOverTime0hours~2hours~20hoursPECD3PE-Cy5CD8PE-Cy7TimeSample

LeftinLightPEPE1423-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第14頁!TandemDyeRemendationsWhenusingAPC-Cy7andPE-Cy7,bewareoffixativeandlightinstabilityissues.IfproblemsarisewhenusingpolystyrenewithAPC-H7,APC-Cy7,orPE-Cy7,tryswitchingtoK-resinorpolycarbonate.PerCP-Cy5.5islesssusceptibletoinstabilitythanAPC-Cy7andPE-Cy7.BDoffersAPC-H7conjugatedantibodiesthataremorestablethanAPC-Cy7conjugatesandhavelessspilloverintotheAPCdetector.1523-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第15頁!NewVioletLaserFluorochromesBDHorizonV500Maximumexcitationat415nm,emissionpeakof500nm.ProvidessignificantlyreducedspilloverintotheFITCchannelparedtoAmCyan—V500haslowexcitationwiththebluelaser.ImprovedstabilitywithfixationparedtoAmCyan.1623-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第16頁!FactorsAffectingReagentPerformanceRelativebrightnessofthefluorochromeNumberoffluorochromesperantibodyExpressionlevelson(orin)thecellsofinterestBackgroundcontributionsSpilloverPhotostabilityReagentenvironmentCytometerconfiguration1723-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第17頁!Non-ConjugatedFluorescenceDyeDetection

BDFACSCantoII4-2-2andBDLSRII4-2-2Detector

RangeVioletLaser

405nmBlueLaser

488nmRedLaser

635nm410–490nmHoechst33342,DAPI,

HorizonVPD450,LIVE/DEADViolet500–560nmLIVE/DEADAqua515–545nmCSFE,GFP,

LIVE/DEADGreen564–606nmPI650–670nmTO-PRO-3670–735nm7-AAD750–810nmLIVE/DEADNear-IRmCherryisbestdetectedwitha561-nmlaseroptionanda590–630detectorrange.1823-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第18頁!SmallOrganicFluorochromesAdvantagesLowmolecularweightEasytoconjugate—directattachmenttofreeaminogroupsonmAbExcellentstabilityExtremelyconsistentemissionspectraDisadvantagesSmallStokesShift(50?100nm)Tendtobelessbright1923-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第19頁!TandemDyeFluorochromesAdvantagesVerylargeStokesShift(150?300nm)Tendtobeverybright—oftenbrighterthanthefluorescentproteindonorDisadvantagesHighmolecularweight(similartofluorescentprotein)Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)Hardertoconjugate(sameasfluorescentprotein)Sometandemshavepoorstability2023-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第20頁!ExcitationandEmissionExcitationwavelengthsdeterminelasersthatcanexcitethefluorochrome.EmissionwavelengthsdeterminefiltersandPMTsthatcanmeasuretheemissionsignal.2123-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第21頁!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-Cy72223-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第22頁!FluorochromeBrightnessThebrightnessofafluorochromedependsontwofactors:MolarExtinctionCoefficient()measureshowwellafluorochromeabsorbsenergy.QuantumYield(Qy)istheratioofphotonsemittedtophotonsabsorbed.Brightness=

x

QyRelativeBrightness=BrightnessofPEBrightness2323-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第23頁!DD=differencebetweenthemediansofthepositiveandnegativepopulationsW

=spread(2xrSD)ofthenegativepopulationStainIndexStainIndex=Stainindexisapracticalwaytocharacterizethebrightnessofamarkerwithrespecttoagivenopticalconfiguration.DWWStainindexcanalsobeusedtocharacterizethesensitivityofafluoresenceparameter.2423-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第24頁!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.2523-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第25頁!CD4StainIndexesAcrossCytometersStainIndexExercise2623-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第26頁!SignificantSpilloverson4-2-2Configuration2723-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第27頁!LostPopulationduetoSpilloverLymphocytesstainedwithCD45FITCandCD4PECD45FITCcausesdimCD4+CD45+

tobedifficulttodistinguishdueto

significantFITCspilloverintoPE.LymphocytesstainedwithCD45PerCPandCD4PECD45PerCPallowsdimCD4+CD45+

tobedistinguishedfrombackground

duetominimalPerCP

spilloverintoPE.2823-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第28頁!TandemDegradation—FalsePositivesFalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7WithCD8APC-Cy7WithoutCD8APC-Cy72923-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第29頁!APC-H7IsMoreStableThanAPC-Cy7ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy73023-13536-00Rev.01流式細胞技術熒光抗體共34頁，您現(xiàn)在瀏覽的是第30頁!NewVioletLaserFluorochromesBDHorizonV450Maximumexcitati