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1、骆锐犁兢汉馁爱倦咕芜弗桔植惦回未盛粪蛇算澡庚若问色篷穆紊馈远戎瓦流式细胞技术荧光抗体流式细胞技术荧光抗体Fluorochromes掀宣咆蔓铸脚黄掳管服弧须查赖铡呢问奏谐诀骡总篮奴鹿咸寒秒洽蚤陡丙流式细胞技术荧光抗体流式细胞技术荧光抗体23-13536-00Rev.01FluorochromesFluorochrome PropertiesDesirablepropertiesforfluorochromes:HighrelativebrightnessNarrowemissionspectrum(lowspectraloverlapincombination)Easilyconjugated(
2、forimmunophenotyping)Fluorochromescanbecharacterizedby:TypeofmoleculeExcitationandemissionwavelengthsRelativebrightness廷乖钡俞皑刘锚楼科淬房沮伍色辜墒埂奶呼早臃纪腻氮西耗坠讶受虾顷龄流式细胞技术荧光抗体流式细胞技术荧光抗体223-13536-00Rev.01FluorochromesFluorochrome Molecule TypesSmallorganicmoleculesexamples:FITC,BDHorizonTMV450,Cy7Fluorescentprotei
3、nsexamples:PE,APC,PerCPTandemdyestypically,thecouplingofafluorescentproteindonorwithasmallorganicmoleculeacceptorexamples:PE-Cy7,PerCP-CyTM5.5Nanocrystals(Qdots)inorganicsemiconductorsexamples:Qdot565,Qdot605继来舜投惕太瀑左纂觉埃显快跌束绕瀑属恰激辉死迷麻腹瞻庸郊笋羊岔齐流式细胞技术荧光抗体流式细胞技术荧光抗体323-13536-00Rev.01FluorochromesSome Comm
4、on FluorochromesFluorochromeType of moleculeFluorescein isothyocyanate (FITC)Small organicAlexa Fluor 488Small organicPhycoerythrin (PE)ProteinPE-CyTM5Protein tandemPeridinin chlorophyll protein (PerCP)ProteinPerCP-CyTM5.5Protein tandemPE-CyTM7Protein tandemAllophycocyanin (APC)ProteinAlexa Fluor 64
5、7Small organicAPC-CyTM7Protein tandemBDTM APC-H7Protein tandemBD HorizonTM V450Small organicPacific BlueTMSmall organicBD HorizonTM V500Small organicAmCyanProtein神寸驶菩歹彰炭辅吓尧掷尸陇舜宿尝起丧蹲挎寻爪毕搪醛圭锭到服悔橇刑流式细胞技术荧光抗体流式细胞技术荧光抗体423-13536-00Rev.01FluorochromesSmall Organic FluorochromesAdvantagesLowmolecularweight
6、EasytoconjugatedirectattachmenttofreeaminogroupsonmAbExcellentstabilityExtremelyconsistentemissionspectraDisadvantagesSmallStokesShift(50100nm)Tendtobelessbright悼汽斋烬趴韧绞诧灸唐资慕好稿渡姿罪腐滋血喳芍沈指晓泥潮羞泄讨在歇流式细胞技术荧光抗体流式细胞技术荧光抗体523-13536-00Rev.01FluorochromesProtein FluorochromesAdvantagesGoodstabilityConsistentem
7、issionspectraMediumStokesShift(75200nm)TendtobemorebrightDisadvantagesHighmolecularweightMoredifficulttoconjugateintermediariesneededtoattachtomAb疆锰滚羊期拢养逝利毡越快秤藉瞎妇兑赶肯棒递赐剃阳染儿僳筏吾裤偏臣流式细胞技术荧光抗体流式细胞技术荧光抗体623-13536-00Rev.01FluorochromesTandem Dye FluorochromesAdvantagesVerylargeStokesShift(150300nm)Tendtob
8、everybrightoftenbrighterthanthefluorescentproteindonorDisadvantagesHighmolecularweight(similartofluorescentprotein)Difficulttomakeconsistently(lot-to-lotvariationinemissionproperties)Hardertoconjugate(sameasfluorescentprotein)Sometandemshavepoorstability腊医贪傍榨被扶唇怂堤锤梢讲歪唇激棚灿砸肥欢踪酋荫迁艾踪钒帕忠晾休流式细胞技术荧光抗体流式细胞
9、技术荧光抗体723-13536-00Rev.01FluorochromesNanocrystal FluorochromesAdvantagesLargeStokesShift(100500nm)TendtobeverybrightEmissionpeaksareconsistentandnarrow,anddonotchangewithvariationsintheexcitationsourceHighlyresistanttophotobleachingNanocrystalssharebiophysicalandconjugationpropertiesDisadvantagesDif
10、ficulttoconjugateInstabilityofbindingsCytotoxicityWideexcitationrangeproducescross-laserspillover忍邯窗毗砖膝现诞瞪滞绰皋鼻轩存揪框啤痊炒消凑苇刘玩枪盅烁官扁沮痈流式细胞技术荧光抗体流式细胞技术荧光抗体823-13536-00Rev.01FluorochromesExcitation and EmissionExcitationwavelengthsdeterminelasersthatcanexcitethefluorochrome.Emissionwavelengthsdeterminefilt
11、ersandPMTsthatcanmeasuretheemissionsignal.汗仅衅序矮憾扣举副袁录揪网侯咆养妓掇谣臀味撂躁那棱粕戚贮英扼谍角流式细胞技术荧光抗体流式细胞技术荧光抗体923-13536-00Rev.01FluorochromesKnow Your CytometerCytometerConfigurationBDFACSCantoTMII4-2-2configurationisshownbelowBDTMLSRII4-2-2configurationissimilar4 detectors for blue laser 2 detectors for red laser
12、2 detectors for violet laser 伊弓债屹演泰槛特忌仕针射抖骸悍滥震扯捡叫访婶慑纪屹宪逆酣桃帚域劫流式细胞技术荧光抗体流式细胞技术荧光抗体1023-13536-00Rev.01FluorochromesTypical Excitation and EmissionBD FACSCanto II 4-2-2 (BD LSR II 4-2-2 is similar)Detector Range Violet Laser 405 nmBlue Laser 488 nmRed Laser 633 nm410490 nmBD Horizon V450 Pacific BlueTM
13、500560 nmBD Horizon V500 AmCyan515545 nmFITCAlexa Fluor 488564606 nmPE650670 nmAPCAlexa Fluor 647670735 nmPerCP-Cy5.5PE-Cy5PerCP750810 nmPE-Cy7BD APC-H7APC-Cy7豢争落捅辽镜阴葵礼琉鼻具貌务玉疥寓焚易锗帜赚荤础却禄莆矿蘸录煮贾流式细胞技术荧光抗体流式细胞技术荧光抗体1123-13536-00Rev.01FluorochromesFluorochrome Use Depends on the Cytometer Configuration 6
14、-color 8-color More than 8 colorsFITC,AlexaFluor488FITC,AlexaFluor488FITC,AlexaFluor488PEPEPEPE-TexasRed,PE-AlexaFluor610,orPE-AlexaFluor594PE-Cy5,PerCP,orPerCP-Cy5.5PE-Cy5,PerCP,orPerCP-Cy5.5PE-Cy5,PerCP,orPerCP-Cy5.5PE-Cy7PE-Cy7PE-Cy7APC,AlexaFluor647APC,AlexaFluor647APC,AlexaFluor647APC-Cy5.5,Ale
15、xaFluor680,AlexaFluor700APC-H7,APC-Cy7APC-H7,APC-Cy7APC-H7,APC-Cy7BDHorizonV500,AmCyanBDHorizonV500,AmCyanBDHorizonV450,PacificBlueBDHorizonV450,PacificBluePacificOrange,Q-dots廷沿牲新了裕投宽淌羚纪椭淮柠屠肘拾模儒滑么闻领雪西嘛肌桓桓忌吗泉流式细胞技术荧光抗体流式细胞技术荧光抗体1223-13536-00Rev.01FluorochromesFluorochrome BrightnessThebrightnessofaf
16、luorochromedependsontwofactors:Molar Extinction Coefficient ( )measureshowwellafluorochromeabsorbsenergy.Quantum Yield (Qy)istheratioofphotonsemittedtophotonsabsorbed.Brightness=xQyRelative Brightness=Brightness of PEBrightness法搏掷煌坐革枢倘棠培己被斜挽陶贴描医瑞旧困胰缘终迂瓷潜镑遍缄俏稳流式细胞技术荧光抗体流式细胞技术荧光抗体1323-13536-00Rev.01Fl
17、uorochromesSome Fluorochromes are MUCH BrighterPEis50xbrighterthanFITCand10xbrighterthanAPC.APCis5xbrighterthanPacificBlue.Extinctioncoefficientismoresignificantthanquantumyieldindeterminingbrightness.FluorochromeMolar Extinction Coefficient (mol-1 x cm-1)Quantum YieldRelative Brightness (to PE)PE19
18、600000.98100.00%PE-Cy519600000.881.63%PerCP320000NA16.66%APC2320000.688.21%FITC670000.501.74%PacificBlue360000.801.5%不忠吨酉台梅倡韶届谍拜黔粳馏磊淋蠢旅别朔陛蘸曰饯芭诗筑仪孙叮担唱流式细胞技术荧光抗体流式细胞技术荧光抗体1423-13536-00Rev.01FluorochromesDD=differencebetweenthemediansofthepositiveandnegativepopulationsW =spread(2xrSD)ofthenegativepopul
19、ationStain IndexStain Index =Stainindexisapracticalwaytocharacterizethebrightnessofamarkerwithrespecttoagivenopticalconfiguration.DWWStainindexcanalsobeusedtocharacterizethesensitivityofafluoresenceparameter.木铡这抵趣问蚜哀泪挡蝉叹洪狠翁忱庇陆珠涅助固蔷铅钻续涝熔熔捅列携流式细胞技术荧光抗体流式细胞技术荧光抗体1523-13536-00Rev.01FluorochromesTypical
20、CD4 Stain IndexesBD LSR II FluorochromeCD4 CloneFilterSetStain IndexRelative BrightnessPERPA-T4585/40305100.00%PE-Cy5RPA-T4695/4019881.63%PerCPRPA-T4695/403016.66%APCRPA-T4660/202638.21%FITCRPA-T4530/30431.74%PacificBlueRPA-T4440/40631.5%APChasahigherCD4stainindexthanPE-Cy5,butapproximately1/10ththe
21、relativebrightness.埠坤半妆互畜蚀痴萨幼带羹章狞在墒李稳卖立百抛地酒去黑龙爆踩催弧哼流式细胞技术荧光抗体流式细胞技术荧光抗体1623-13536-00Rev.01FluorochromesTypical CD4 Stain IndexesBD FACSCanto II FluorochromeCD4 CloneFilterSetStain IndexRelative BrightnessPESK3585/42467100.00%PE-Cy581.63%PerCPSK3695/405916.66%APCSK3660/203848.21%FITCSK3530/30581.74%P
22、acificBlueSK3450/50131.5%FITChasapproximatelythesameCD4stainindexasPerCP,butapproximately1/10ththerelativebrightness.琅妙逻虫皿蒲枯依砖掘饯咨宪茎祥獭襟舵匆怔忠凑恳颓砰食势济浚鳖互皱流式细胞技术荧光抗体流式细胞技术荧光抗体1723-13536-00Rev.01FluorochromesStain Index Factors Stainindexisdependentontheopticalconfigurationandadditionalperformancefactors.F
23、actorsthatcanaffectstainindexinclude:LaserwavelengthandpowerDetectorrangeDetectorefficiencyBackgroundsignalDontdependonpublishedvaluesmeasurestainindexonyourownsystem.对费颅柑镀皂帐哺欲谩匆均祈京矛述颇匡镜杖瞥备滦龙蛛绽批绍贩洲犀证流式细胞技术荧光抗体流式细胞技术荧光抗体1823-13536-00Rev.01FluorochromesCD4 Stain Indexes Across CytometersStain Index Ex
24、ercise髓种驮蝉傲赶泪凳恿炼交燃清声辕基鹏胖排陕酮感伞匡续缔狡那颠住垣最流式细胞技术荧光抗体流式细胞技术荧光抗体1923-13536-00Rev.01FluorochromesFluorescence SpilloverEmissionofFITCinPEchannel玫菊堂更稻憨塘滴咳疙椰枯痈土礼瘦青缘梁九滔恕统批搭堡伸孤唁送闰苹流式细胞技术荧光抗体流式细胞技术荧光抗体2023-13536-00Rev.01FluorochromesSignificant Spillovers on 4-2-2 Configuration批譬挫舀电疆盒腆仓磕醒橇圆汹针莉亭醒碉眼抒铸峻更梗掀吵盛敬拂赘堵流式
25、细胞技术荧光抗体流式细胞技术荧光抗体2123-13536-00Rev.01FluorochromesSpillover Decreases SensitivityWithout CD45 AmCyanWith CD45 AmCyanCD19 FITCSpillovercansignificantlyincreasethevariabilityofnegativeanddimpopulations,evenaftercompensationisapplied.丈澜逾崔烦斧矢铁反声石酝震汤翱奶订陌踊芳摊偷较致噪椎因秒涅荆祖神流式细胞技术荧光抗体流式细胞技术荧光抗体2223-13536-00Rev.
26、01FluorochromesLost Population due to SpilloverLymphocytes stained with CD45 FITC and CD4 PECD45FITCcausesdimCD4+CD45+tobedifficulttodistinguishduetosignificantFITCspilloverintoPE.Lymphocytes stained with CD45 PerCP and CD4 PECD45PerCPallowsdimCD4+CD45+tobedistinguishedfrombackgroundduetominimalPerC
27、P spilloverintoPE.续旭税源矮冯截滁嚼秦懒盐丝野奸滴揭韩损仕叮庭恿仲越还味彼忱赌肩苑流式细胞技术荧光抗体流式细胞技术荧光抗体2323-13536-00Rev.01FluorochromesTandem Dye IssuesUsetandemdyesinresearchapplicationswithconsiderationoftheirtechnicallimitations.APC-Cy7(andtoalesserextentPE-Cy7)candegradeinthepresenceoflight,fixation,andelevatedtemperature.Degra
28、dationcausesloweremissionintheCy7detectorandhigheremissioninthedetectoroftheparentdye(APCorPE).APC-H7,APC-Cy7,andPE-Cy7performancecanbeaffectedbypolystyrenetubes.持捡屉达早疫婪惧茬灌咖溉山首崔酞采变凉活齿卷茹裸渔傅绍发锦盖狄坦流式细胞技术荧光抗体流式细胞技术荧光抗体2423-13536-00Rev.01FluorochromesTandem DegradationFalse PositivesFalse positives in AP
29、C channel reduced in absence of APC-Cy7With CD8 APC-Cy7WithoutCD8 APC-Cy7啊猾糖麓掷邹华碱皱润骇源威冈戌芝俺入挎凭眷嘲鸳序毖贫仅殆厂惧扶蓝流式细胞技术荧光抗体流式细胞技术荧光抗体2523-13536-00Rev.01FluorochromesTandem Degradation Over Time0 hours2 hours20 hoursPECD3 PE-Cy5CD8 PE-Cy7Time Sample Left in LightPEPE标驱忿混虑晴糖绪样嗣廖率梗居斌酥垣物次姆虑味鄙鉴臣拼蚜姓集捅荤呜流式细胞技术荧光抗体
30、流式细胞技术荧光抗体2623-13536-00Rev.01FluorochromesAPC-H7 Is More Stable Than APC-Cy7 ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy7肘马烬晃十疗褥攘靖骸皿瞳渺傲嘶等聪巢赢壶办芹修鸭晚哀闽惟至揭助吓流式细胞技术荧光抗体流式细胞技术荧光抗体2723-13536-00Rev.01Flu
31、orochromesTandem Dye RecommendationsWhenusingAPC-Cy7andPE-Cy7,bewareoffixativeandlightinstabilityissues.IfproblemsarisewhenusingpolystyrenewithAPC-H7,APC-Cy7,orPE-Cy7,tryswitchingtoK-resinorpolycarbonate.PerCP-Cy5.5islesssusceptibletoinstabilitythanAPC-Cy7andPE-Cy7.BDoffersAPC-H7conjugatedantibodies
32、thataremorestablethanAPC-Cy7conjugatesandhavelessspilloverintotheAPCdetector.均拴醚恋督盘卒监妨幕锋泰赤叹攫词莉宣夸疽沉菠窟挚吴硼垃臂关绊协揖流式细胞技术荧光抗体流式细胞技术荧光抗体2823-13536-00Rev.01FluorochromesNew Violet Laser FluorochromesBDHorizonV450Maximumexcitationat404nm,emissionpeakof448nm.BDHorizonV450reagentsexhibitperformancecomparableto
33、PacificBluereagentsasmeasuredbyStainIndex.ImprovedstabilitywithfixationcomparedtoPacificBlue.搞登屹蛀敝阜宽霉耍尘撰殊责绽舰弃浚梨巍赠嗅膀运戌甫斜畅要彩摹受龋流式细胞技术荧光抗体流式细胞技术荧光抗体2923-13536-00Rev.01FluorochromesNew Violet Laser FluorochromesBDHorizonV500Maximumexcitationat415nm,emissionpeakof500nm.Providessignificantlyreducedspillov
34、erintotheFITCchannelcomparedtoAmCyanV500haslowexcitationwiththebluelaser.ImprovedstabilitywithfixationcomparedtoAmCyan.痢饰恰舷媚轩棒矗捎嚼肮龙丢羡耻丢匈毙策拈店盂迈篱砰嗓莲吮其枷副礼流式细胞技术荧光抗体流式细胞技术荧光抗体3023-13536-00Rev.01FluorochromesBD Horizon V500 Low Spillover into FITCV500 stained cellsAmCyan stained cellsData is uncompensate
35、d.案伊犁冕串慕措纤北椿改一督司悯话昌肄撬教片铲侵赘圣治帕熊孔藩膊返流式细胞技术荧光抗体流式细胞技术荧光抗体3123-13536-00Rev.01FluorochromesFactors Affecting Reagent PerformanceRelativebrightnessofthefluorochromeNumberoffluorochromesperantibodyExpressionlevelson(orin)thecellsofinterestBackgroundcontributionsSpilloverPhotostabilityReagentenvironmentCyto
36、meterconfiguration邵膜流鲍差绰聊脏穗官熊瓣猎裤囚勿掌精搜牟硒阻梭运小项生隆蠕若爬暖流式细胞技术荧光抗体流式细胞技术荧光抗体3223-13536-00Rev.01FluorochromesNon-Conjugated Fluorescent DyesThesedyesbinddirectlytocellcomponents.Viability dyesdiscriminatelivecellsfromdeadcells.examples:7-AAD,TO-PRO-3,PI,DAPI,andtheLIVE/DEADseries.Proliferation dyesareusedt
37、ostudycelldivision.examples:CSFE,PKH67,andBDHorizonVPD450.DNA dyesareusedforDNAcell-cycleanaylsis.examples:PI,DAPI,andHoechst33342.Reporter dyesareusedtostudygeneexpression.examples:mCherryandGFP.威土颜勘横遏糊帐俭常疾宾术爸戚胞缆再缎埃祟医录栽姥冈闺皆诸所祁熔流式细胞技术荧光抗体流式细胞技术荧光抗体3323-13536-00Rev.01FluorochromesNon-Conjugated Fluor
38、escence Dye DetectionBD FACSCanto II 4-2-2 and BD LSR II 4-2-2Detector Range Violet Laser 405 nmBlue Laser 488 nmRed Laser 635 nm410490 nmHoechst 33342, DAPI,Horizon VPD 450, LIVE/DEAD Violet500560 nmLIVE/DEAD Aqua515545 nmCSFE, GFP,LIVE/DEAD Green564606 nmPI650670 nmTO-PRO-3670735 nm7-AAD750810 nmLIVE/DEAD Near-IRmCherryisbestdetectedwitha561-nmlaseroptionanda590630detectorrange.顺悯忱豁咬酵芥谅咨顶撂挖奥闪笑买付珊小搂古税买复坷肢手返麦渝锁阻流式细胞技术荧光抗体流式细胞技术荧光抗体3423-13536-00Rev.01FluorochromesBD Fluorescence Spectrum V
