11月成都熒光標(biāo)記與檢測(cè)細(xì)胞結(jié)構(gòu)健康

AdvancesinFluorescenceLabeling&DetectionStrategiesforCellularAdvancesinFluorescenceLabeling&DetectionStrategiesforCellularAnalysis——熒光標(biāo)記與檢測(cè)，細(xì)胞結(jié)構(gòu)與健康鄭博NewBusinessDevelopmentManagerLifeTechnologies?Proprietary|12014，Life2014，LifeSciencesSolutionsGroup（LSG），賽默飛世爾科技內(nèi)部五大運(yùn)營商之一1962GIBCONYLifeTechnologies?Proprietary|28個(gè)值得信賴的品牌/超過60萬次8個(gè)值得信賴的品牌/超過60萬次植根于科學(xué)由科學(xué)家創(chuàng)造以創(chuàng)新驅(qū)動(dòng)LifeTechnologies?Proprietary|3MolecularProbes的歷史1967197819862003開創(chuàng)性的FRET……搬去特克薩斯州遷往俄勒岡州Eugene30年來并成立新MolecularProbes的歷史1967197819862003開創(chuàng)性的FRET……搬去特克薩斯州遷往俄勒岡州Eugene30年來并成立新OregonGreen?廣泛引用：30,000篇物旗艦產(chǎn)標(biāo)準(zhǔn)（如AlexaFluor?染料）始人Richard和RosariaHaugland博士頓開業(yè)遷往俄勒岡州Junction市更好的……“細(xì)胞、孔和凝膠”早期試劑197519821995LifeTechnologies?Proprietary|44Eugene,OregonLifeTechnologies?Eugene,OregonLifeTechnologies?Proprietary|55InvitrogenProprietary&劉翔在尤金贏得110M高欄金牌劉翔在尤金贏得110M高欄金牌?sLiuXiangcelebratesafterwinningthegoldmedalintheMen's110mHurdlesduringthePrefountainClassicGameinEugene,Oregon.photo:AP/DavidGuttenfelderLifeTechnologies?Proprietary|6LifeTechnologiesMolecularLifeTechnologiesMolecularProbes?LifeTechnologies?Proprietary|77細(xì)胞分析是生命科學(xué)研究的基礎(chǔ)熒光技術(shù)是進(jìn)行細(xì)胞分析的基礎(chǔ)cancergeneticsbiochemistryLifeTechnologies?Proprietary細(xì)胞分析是生命科學(xué)研究的基礎(chǔ)熒光技術(shù)是進(jìn)行細(xì)胞分析的基礎(chǔ)cancergeneticsbiochemistryLifeTechnologies?Proprietary|88FluorescenceTechnologyinBiologicalResearchHTSMultiplexedFluorescenceTechnologyinBiologicalResearchHTSMultiplexedAssaysHighContentScreeningFlowCytometrySmallAnimalInvivoImagingMicroscopyGenomicsProteomicsLifeTechnologies?Proprietary|9MolecularProbes:CellularAnalysisPortfolioAssays,Reagents&AntibodiesHCA/HCSSystemsFlowCytometryMolecularProbes:CellularAnalysisPortfolioAssays,Reagents&AntibodiesHCA/HCSSystemsFlowCytometry?InnovativeFlowCytometersusingCellpopulationanalysis??FullRangeofsimple,intuitiveinstrumentsCellcountsandcellhealthanalysisFluorescenceandBrightfieldimagingAutomatedimagingandanalysisatindividualcellsandcellpopulationlevelReagentsforLabeling,Imaging,andFlowCytometry10,000’sofkits,antibodiesanddyes???LifeTechnologies?Proprietary|10MolecularProbes?的MolecularProbes?的占?xì)W美免疫熒光試劑市場份額的40.7%第三方市場調(diào)研數(shù)據(jù)LifeTechnologies?Proprietary|11MolecularProbesMolecularProbes?熒光檢測(cè)及免疫熒光標(biāo)記LifeTechnologies?Proprietary|12Agenda2和解決方案4.熒光標(biāo)記中的新“”及應(yīng)用LifeTechnologies?Agenda2和解決方案4.熒光標(biāo)記中的新“”及應(yīng)用LifeTechnologies?Proprietary|13Agenda2.熒光檢測(cè)中常見問題和解決方案4.熒光標(biāo)記中的新“”和應(yīng)用LifeAgenda2.熒光檢測(cè)中常見問題和解決方案4.熒光標(biāo)記中的新“”和應(yīng)用LifeTechnologies?Proprietary|14為什么要進(jìn)為什么要進(jìn)行熒光顯微成像？對(duì)比！DIC圖像熒光圖像LifeTechnologies?Proprietary|15兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔免兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔免兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔LifeTechnologies?Proprietary|16兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔免兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔免兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔兔LifeTechnologies?Proprietary|17熒光標(biāo)記&檢測(cè)熒光紫外到紅外活細(xì)胞和/或固定細(xì)胞光不穩(wěn)定到近紅外熒光標(biāo)記&檢測(cè)熒光紫外到紅外活細(xì)胞和/或固定細(xì)胞光不穩(wěn)定到近紅外光不穩(wěn)定綠色到紅外活細(xì)胞和/或固定細(xì)胞光穩(wěn)定性很好抗體&鏈霉親和素結(jié)合物細(xì)胞示蹤離子&電壓指示劑細(xì)胞器蛋白表達(dá)有功能的“生物傳感器”細(xì)胞器靶定抗體&鏈霉親和素結(jié)合物細(xì)胞示蹤成像電子顯微鏡LifeTechnologies?Proprietary|18熒光納米晶體熒光蛋白常見的熒光（EB）（FITC常見的熒光（EB）（FITC）LifeTechnologies?Proprietary|19PhenolphthaleinFlexible,non-fluorescentLifeLifeTechnologies?Proprietary|20小結(jié)：Life小結(jié)：LifeTechnologies?Proprietary|21可見光光譜Life可見光光譜LifeTechnologies?Proprietary|22ExcitationEmissionLifeTechnologies?ExcitationEmissionLifeTechnologies?Proprietary|23LifeTechnologies?LifeTechnologies?Proprietary|24LifeLifeTechnologies?Proprietary|25HowtoChoosetheOptimalFluorophoreKnowHowtoChoosetheOptimalFluorophoreKnowTheInstrument’sLimitations– Excitationsource(s)anddetectionfiltersKnowTheFluorophore’sLimitationsExcitation/emissionExtinctioncoefficientaKnowtheTargetUsetheSpectraviewerLifeTechnologies?Proprietary|26pro/spectraviewer2LifeLifeTechnologies?Proprietary|27LifeLifeTechnologies?Proprietary|28LifeLifeTechnologies?Proprietary|29LifeLifeTechnologies?Proprietary|30LifeLifeTechnologies?Proprietary|31LifeLifeTechnologies?Proprietary|32LifeLifeTechnologies?Proprietary|33LifeLifeTechnologies?Proprietary|34LifeLifeTechnologies?Proprietary|35LifeLifeTechnologies?Proprietary|36LifeLifeTechnologies?Proprietary|37LifeLifeTechnologies?Proprietary|38LifeLifeTechnologies?Proprietary|39HowHowtoSelecttheRightCombinationofReagentsforYourMulticolorImagingandFlowAssay如何選擇最佳熒光試劑組合SelectthephotostabledyesUseLifeTechnologies’“FluorescenceSpectraViewer”tocheckthespectralpropertyofreagent(s)tobeused(notjustex/em,needtoreviewthewholespectrum)SelectdyesthatnotspilloverlargelyintootherchannelsSelectdyesthathavelessspectraloverlapSelectdyesthatcanbeexcitedwithdifferentlasersSelectbrightfluorophoreforweakermarkerChangefilter(s)ifneededLifeTechnologies?Proprietary|40Agenda12.熒光檢測(cè)中的主要&解決方案4”和應(yīng)用LifeAgenda12.熒光檢測(cè)中的主要&解決方案4”和應(yīng)用LifeTechnologies?Proprietary|41問題：或者…或者LifeTechnologies?Proprietary問題：或者…或者LifeTechnologies?Proprietary|42光漂白是對(duì)的不可逆Photo-Bleach破壞，因激發(fā)過程中光漂白是對(duì)的不可逆Photo-Bleach破壞，因激發(fā)過程中產(chǎn)生自由基.LifeTechnologies?Proprietary|431：問題：或者…或者LifeTechnologies?Proprietary1：問題：或者…或者LifeTechnologies?Proprietary|44AlexaFluor?–內(nèi)在光穩(wěn)定性第二代vs.傳統(tǒng)更亮、更穩(wěn)定AlexaFluor?–內(nèi)在光穩(wěn)定性第二代vs.傳統(tǒng)更亮、更穩(wěn)定LifeTechnologies?Proprietary|45AlexaFluor?SecondaryAntibodyConjugates:TheAlexaFluor?SecondaryAntibodyConjugates:TheMostWidelyPublished多色選擇更亮，耐光淬滅性更強(qiáng)LifeTechnologies?Proprietary|47Qdot?納米晶體：相Qdot?納米晶體：相對(duì)大小與蛋白大小相當(dāng)…非常明亮且光穩(wěn)定LifeTechnologies?Proprietary|48Qdot?納米晶體：熒Qdot?納米晶體：熒光光譜紫色激發(fā)發(fā)射范圍寬最佳的紫色激發(fā)，適合所有發(fā)射光譜的多種Qdot?探針，讓多重分析變得簡單LifeTechnologies?Proprietary|49Qdot?亮度&光穩(wěn)定性促成了高靈敏度Qdot?655結(jié)合物0.019sCy5dye結(jié)Qdot?亮度&光穩(wěn)定性促成了高靈敏度Qdot?655結(jié)合物0.019sCy5dye結(jié)合物1.22s60XSK-BR-3細(xì)胞：Her2/neu表達(dá)高7X22X0.44s8.12sMDA-MB-231細(xì)胞：Her2/neu表達(dá)低ImagesbyXingyongWu亮度不夠時(shí)，Qdot?探針可檢測(cè)稀有目標(biāo)。50當(dāng)Technologies?Proprietary|Anti-Her2/neu+anti-mouseIgGconjugatesQdot?納米晶體用于免疫標(biāo)記Qdot?655/anti-tubulinQdot?納米晶體用于免疫標(biāo)記Qdot?655/anti-tubulinIgGconjugateSlidesCourtesyofTomDeerinckandMarkEllisman,UCSD適用于熒光和電子顯微鏡LifeTechnologies?Proprietary|51用Qdot?納AlexaFluor?進(jìn)用Qdot?納AlexaFluor?進(jìn)行小動(dòng)物成像Anti-CEAIgG-AlexaFluor?680conjugate復(fù)合物Qtracker?800non-targetedquantumdotsLifeTechnologies?Proprietary|52Qdot?技術(shù)的廣泛應(yīng)用525545565585605625655臨床前成像手術(shù)Qdot?技術(shù)的廣泛應(yīng)用525545565585605625655臨床前成像手術(shù)指導(dǎo)受體示蹤試劑盒病理學(xué)/免疫組化流式細(xì)胞儀活細(xì)胞示蹤(Qtracker?試劑盒)固定細(xì)胞顯微鏡觀察LifeTechnologies?Proprietary|53細(xì)胞示蹤的、量子點(diǎn)和熒光蛋白短期示蹤劑CalceinAMCellMask?質(zhì)膜長期示蹤細(xì)胞示蹤的、量子點(diǎn)和熒光蛋白短期示蹤劑CalceinAMCellMask?質(zhì)膜長期示蹤劑CellTrackerCellTraceQtracker?探針熒光蛋白*反應(yīng)性基團(tuán)決定了長期vs.短期標(biāo)記的適用性AM乙酰氧基甲酯：掩蓋了使探針可透過活細(xì)胞的電荷；基于電荷的細(xì)胞內(nèi)滯留CM=氯甲基：巰基反應(yīng)基團(tuán)通常導(dǎo)致可固定的標(biāo)記SE琥珀酰亞胺酯：胺反應(yīng)基團(tuán)，與賴氨酸上的伯胺結(jié)合LifeTechnologies?Proprietary|541：問題：或者…或者LifeTechnologies?Proprietary1：問題：或者…或者LifeTechnologies?Proprietary|55固定細(xì)胞中的抗淬滅保護(hù)固定細(xì)胞中的抗淬滅保護(hù)用ProLong?Gold抗淬滅劑封片僅用緩沖液封片更強(qiáng)的初始強(qiáng)度和對(duì)光漂白的保護(hù)ProLongGold?SlowFade?GoldLifeTechnologies?Proprietary|56AntifadeMountingAgents抗淬滅封片劑PProLong?Gold—AntifadeMountingAgents抗淬滅封片劑PProLong?Gold—polymerizingSlowFade?Gold—non-polymerizingLifeTechnologies?Proprietary|57Problem:fluorescenceofasampledecreaseorlostovertimeaftermounting MountingsolutionsweofferWithroLong?mountingmountingQdot?mountingIHCmountingDABandBCIP/NBTWithout?ProLongGoldantifadeAntifadereagentSlowFade?GoldAntifadereagentSolution:AntisampleClearmountMountingSolutionreagentsprotectQmount?Qdot?mountingmedia&extendthefluorescHistomountMountingSolutionenceofaProlong?Gold抗淬Prolong?Gold抗淬滅劑：整個(gè)光譜的保護(hù)數(shù)值表示為30秒連續(xù)光照后的熒光更強(qiáng)的初始強(qiáng)度和對(duì)光漂白的保護(hù)LifeTechnologies?Proprietary|58Agenda1.熒光檢測(cè)流程及相關(guān)2&解決方案4”LifeAgenda1.熒光檢測(cè)流程及相關(guān)2&解決方案4”LifeTechnologies?Proprietary|59成像實(shí)驗(yàn)流程鏡細(xì)胞核復(fù)染&封片鏡固定細(xì)胞固定/通透洗&封閉大量樣品分析成像實(shí)驗(yàn)流程鏡細(xì)胞核復(fù)染&封片鏡固定細(xì)胞固定/通透洗&封閉大量樣品分析長期保存基于抗體的檢測(cè)抗體固定細(xì)胞Qdot偶聯(lián)物細(xì)胞形態(tài)及研究LifeTechnologies?Proprietary|60非損傷性實(shí)時(shí)觀測(cè)細(xì)胞生理學(xué)免疫熒光檢測(cè)流程對(duì)于整個(gè)免疫熒光檢測(cè)流程,我們都有很好的提供LifeTechnologies?免疫熒光檢測(cè)流程對(duì)于整個(gè)免疫熒光檢測(cè)流程,我們都有很好的提供LifeTechnologies?Proprietary|61專適于成像的遇到的問題：固定、透化、封閉試劑盒需訂購多個(gè)不同的試劑；需配制相關(guān)的固定、通透、封閉和洗液；需保存在不同的條件；比較繁瑣；處理效果？解決方案：Image-iT?固定/透化試劑盒（專適于成像的遇到的問題：固定、透化、封閉試劑盒需訂購多個(gè)不同的試劑；需配制相關(guān)的固定、通透、封閉和洗液；需保存在不同的條件；比較繁瑣；處理效果？解決方案：Image-iT?固定/透化試劑盒（R37602）試劑盒包括以下組分，處理后可立即進(jìn)行免疫染色。固定液通透液洗液封閉液關(guān)鍵賣點(diǎn)：1x即用型試劑盒，可固定、透化并洗滌細(xì)胞；?無需新鮮的固定劑；室溫保存（18個(gè)月）；優(yōu)化的配方有助于減少背景染色，提高圖像的質(zhì)量；適用于熒光蛋白、62、抗體和Click-iT?試劑。Technologies?Proprietary|免疫熒光檢測(cè)流程非特異背景的來源自發(fā)熒光(細(xì)胞或組織的內(nèi)在熒光)免疫熒光檢測(cè)流程非特異背景的來源自發(fā)熒光(細(xì)胞或組織的內(nèi)在熒光)減少策略遠(yuǎn)紅外光譜,如AlexaFluor647放大特異信號(hào),TSA蛋白非特異結(jié)合使用封閉液如BSA或血清的結(jié)合非特異結(jié)合-電荷通常的封閉液如血清或BSA不起作用LifeTechnologies?Proprietary|63非特異的背景Life非特異的背景LifeTechnologies?Proprietary|64Blockingnon-specificbindingisessentialBlockingnon-specificbindingisessential–BlockAid?isbestHomebrewHomebrewHumanC-terminusoftalinThesesampleswereincubatedwithfluorescentsecondaryantibodiesandshownonspecificbinding.LifeTechnologies?Proprietary|65Non-specificfluorescenceBlockAid?-CrispBlockAid?-CrispBeautifulSpecificFluorescence?????Readytouse-nomixingorformulationCustomersusewitheveryantibody-baseddetectionMostcustomershomebrewwithserum(e.g,BSA)BlockAid?pricepersamplecomparablewithhomebrewsolutionUniversal-don‘tneeddifferentformulationsfordifferentantibodyspeciescombinationsCatalogNumber:B10710?LifeTechnologies?Proprietary|66為什么需要Image-iT?FXSignalEnhancer?問題:熒光基團(tuán)可能結(jié)合在一些細(xì)胞為什么需要Image-iT?FXSignalEnhancer?問題:熒光基團(tuán)可能結(jié)合在一些細(xì)胞結(jié)構(gòu)上,造成很高的熒光背景。通常的封閉液如血清或BSA不起作用解決方案Image-IT?FXSignalEnhancer可以抑制由熒光來的非特異性背景。帶Image-iTFX?SignalEnhancerUntreatedEndothelialCellsMouseHippocampusAlexaFluor?488antibodyNoprimaryantibodyLifeTechnologies?Proprietary|67我在這里呢。通過選擇性地抑制背景干擾，可以使特定的信號(hào)變得更明顯。Image-iT?FX我在這里呢。通過選擇性地抑制背景干擾，可以使特定的信號(hào)變得更明顯。Image-iT?FXSignalEnhancerLifeTechnologies?Proprietary|68免疫熒光檢測(cè)流程Abfinity?binantantibodiesLife免疫熒光檢測(cè)流程Abfinity?binantantibodiesLifeTechnologies?Proprietary|69免疫熒光檢測(cè)流程靶：黑色一抗：二抗：紅色：黃色LifeTechnologies?免疫熒光檢測(cè)流程靶：黑色一抗：二抗：紅色：黃色LifeTechnologies?Proprietary|70常用的免疫檢測(cè)方法Life常用的免疫檢測(cè)方法LifeTechnologies?Proprietary|71DetectDetectspecificproteins?OnlyLifeTechnologiesDetectDetectspecificproteins?OnlyLifeTechnologieshasthesuiteofdetectiontoolsforanyleveloftargetabundancePre-labeledprimariesorAntibody/proteinlabelingkitswithAlexaFluor?dyesSecondaryantibodieswithAlexaFluor?labels–fastlabelingandbrightsignalsStreptavidinconjugateswithAlexaFluor?labels-whenyouneedtoampupthesignalTSAamplificationkitsTechnologies?Proprietary|72AntibodyDetection101Usetherighttoolsforbrightnessandsensitivity:ConjugatedprimaryantibodiesandtargetingproteinsAntibodyDetection101Usetherighttoolsforbrightnessandsensitivity:Conjugatedprimaryantibodiesandtargetingproteins–fastestwaytodetect?SecondaryantibodieswithAlexaFluor?andQdot?labels–fastandbrightStreptavidinconjugatesprovidewavelengthselectionandbrightness-whenyouneedtoampupthesignalOnlyLifeTechnologiesoffersacompleteportfolioofdetectiontoolsforoptimumresultswithanyabundanceoftarget?providesultimatesensitivity–brightnessforevenfortheleastabundanttargetsLifeTechnologies?Proprietary|73免疫細(xì)胞化學(xué)和免疫免疫細(xì)胞化學(xué)和免疫組織化學(xué)的常用標(biāo)記和檢測(cè)策略LifeTechnologies?Proprietary|74問題:多個(gè)同型且宿主相同的一抗的檢測(cè)實(shí)驗(yàn)中特異檢測(cè)目標(biāo)，但是……常常只有問題:多個(gè)同型且宿主相同的一抗的檢測(cè)實(shí)驗(yàn)中特異檢測(cè)目標(biāo)，但是……常常只有同一個(gè)宿主的抗體不同亞型的二抗？如果亞型也相同呢？！復(fù)雜且耗時(shí)的三重標(biāo)記步驟或……一抗結(jié)合物，但是……反應(yīng)性會(huì)標(biāo)記IgG的可變區(qū)，從而會(huì)影響抗體與目標(biāo)的親和力的載體蛋白（eg.BSA）解決方案：Zenon?技術(shù)Apex技術(shù)LifeTechnologies?Proprietary|75解決方案：Zenon?技術(shù)解決方案：Zenon?技術(shù)（非共價(jià)結(jié)合）多個(gè)相同宿主一抗的Fc-特異且快速的免疫標(biāo)記LifeTechnologies?Proprietary|76解決方案：APEX?抗體標(biāo)記試劑盒(共價(jià)結(jié)合）解決方案：APEX?抗體標(biāo)記試劑盒(共價(jià)結(jié)合）APEX?標(biāo)記要求：10-20gIgG抗體標(biāo)(200體積)~15分鐘“辛勤勞動(dòng)‖(總共2.5小時(shí))通過移液器，洗脫洗滌緩沖液標(biāo)記的IgG通過移液器，上樣APEX?標(biāo)記吸頭上樣IgG抗體熒光標(biāo)記洗滌緩沖液多個(gè)步驟：上樣或洗脫IgGAPEX?抗體標(biāo)記吸頭輕松的一抗共價(jià)標(biāo)記LifeTechnologies?Proprietary|78抗體標(biāo)記和蛋白修飾:蛋白的摩爾比被稱為DOL（degreeoflabeling標(biāo)記程度）DOL的優(yōu)化很重要！剛抗體標(biāo)記和蛋白修飾:蛋白的摩爾比被稱為DOL（degreeoflabeling標(biāo)記程度）DOL的優(yōu)化很重要！剛好(DOL=2)太多了(DOL=6)抗原結(jié)合位點(diǎn)修飾&隨后的功能損失2+:蛋白獲得了信號(hào)放大之間&熒光不增強(qiáng)無之間相互作用&隨后的自我淬滅因蛋白表面而導(dǎo)致沉淀抗原結(jié)合位點(diǎn)無修飾LifeTechnologies?Proprietary|79LabelingOptionsfromMolecularProbesSometimesyoucannotpurchasetheconjugaterequired.AvarietyLabelingOptionsfromMolecularProbesSometimesyoucannotpurchasetheconjugaterequired.AvarietyofkitsareavailablefromMolecularProbes?forlabelingAbsandproteinstomeetyourlabelingneeds.LabelKitAvailableProductNotes<1–20gLabelingTechnologyNon-covalentlabelingRapid,versatileandreliable10–20gAPEXAbLabelingKits-CovalentlabelingofIgGEfficient,convenientandreliableLabelinpresenceofstabilizingproteinsandamine-containingbuffers10–100gMicroscaleproteinlabeling-LabelingpurifiedproteinwithMW(12and150kDa)100gMonoclonalAbLabelingkit-DirectlylabelingmAborpAb1mgProteinLabelingKits-LabellargeramountsofIgG-2hrlabelingLiTcholgie?rorery0aidAblaigKits-Rigorouslycontrolleddegreeoflabelinglabelingwithnear-IRAlexaFluor?dye免疫細(xì)胞化學(xué)和免疫免疫細(xì)胞化學(xué)和免疫組織化學(xué)的標(biāo)記和檢測(cè)策略LifeTechnologies?Proprietary|81問題:二抗檢測(cè)有時(shí)不夠靈敏，特別是稀有目標(biāo)LabelLabelLabel解決方案:酪胺信號(hào)放大(TSA?)LifeTechnologies?Proprietary|82熒光基團(tuán)的共價(jià)沉積問題:二抗檢測(cè)有時(shí)不夠靈敏，特別是稀有目標(biāo)LabelLabelLabel解決方案:酪胺信號(hào)放大(TSA?)LifeTechnologies?Proprietary|82熒光基團(tuán)的共價(jià)沉積酪胺信號(hào)放大(TSA?)如何在免疫熒光標(biāo)記策略中起作用？350488532546568594647Technologies?Proprietary|83DyeDye酪胺信號(hào)放大(TSA?)如何在免疫熒光標(biāo)記策略中起作用？350488532546568594647Technologies?Proprietary|83DyeDye免疫組化和TSA?免疫組化和TSA?anti–GFAPAlexaFluor?488Goatanti–MouseIgGAlexaFluor?488Tyramide200msec40msec小鼠小腦中的伯格曼膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞TSA?超靈敏檢測(cè)，否則信息會(huì)丟失在組織樣品的高熒光背景之中LifeTechnologies?Proprietary|84熒光二抗選擇指南第一步:根據(jù)一抗的物種來源。例如:如果一抗是小鼠來源的,就找“anti-mouse”第二步:(IgA,IgD,IgE,IgGIgG1,IgG2,熒光二抗選擇指南第一步:根據(jù)一抗的物種來源。例如:如果一抗是小鼠來源的,就找“anti-mouse”第二步:(IgA,IgD,IgE,IgGIgG1,IgG2,IgG2a,2b,2c,IgG3,IgG4,IgM,IgY,Kappalightchain,Lambdalightchain)第三步:選擇抗體的形式Wholeantibody或F(ab)2片段抗體。第四步:選擇耦聯(lián)的的熒光素。AlexaFluor?488F(ab')2fragmentofrabbitanti-mouseIgG(H+L)LifeTechnologies?Proprietary|85TypeProsConsWholeIgGStrongestsignal,inexpensivePossiblecross-reactivityF(ab)2WouldnotinteractwithF(c)receptorsLowersignalduetofewerdyemoleculesSpecificIgG1,IgG2a,IgG2b,IgG3,IgMExpensive,mustknowsubtypeofprimary熒光顏色之“解”EmissionBluegreenorange/redfarred350400450500550600650700LifeTechnologies?Proprietary|86DAPI/UVFITCTRITCFARREDAlexaFluor350 AlexaFluor熒光顏色之“解”EmissionBluegreenorange/redfarred350400450500550600650700LifeTechnologies?Proprietary|86DAPI/UVFITCTRITCFARREDAlexaFluor350 AlexaFluor488Coumarin,AMCA Fluorescein(FITC)Cy2AlexaFluor AlexaFluor647555 Cy5,APCRhodamine,TAMRA,TRITCCy3 AlexaFluor594TexasRed,Cy3.5365nm 407nm 488nm 543nm 633nm傳統(tǒng)vs第二代傳統(tǒng)第二代歷史悠久。性能明顯改善。– Alexa傳統(tǒng)vs第二代傳統(tǒng)第二代歷史悠久。性能明顯改善。– AlexaFluor?Dyes真正的二代。多色選擇更亮，耐光淬滅性更強(qiáng)LifeTechnologies?Proprietary|87AlexaFluor?–內(nèi)在光穩(wěn)定性第二代vs.傳統(tǒng)更亮、更穩(wěn)定AlexaFluor?–內(nèi)在光穩(wěn)定性第二代vs.傳統(tǒng)更亮、更穩(wěn)定LifeTechnologies?Proprietary|88免疫熒光檢測(cè)流程核酸LifeTechnologies?免疫熒光檢測(cè)流程核酸LifeTechnologies?Proprietary|89AntifadeMountingAgents抗淬滅封片劑FITCdyeconjugatewithProLong?AntifadeMountingAgents抗淬滅封片劑FITCdyeconjugatewithProLong?Gold(P36930)t=0sect=5sect=10sect=20sect=15secFITCdyeconjugateinPBSLifeTechnologies?Proprietary|90ProLong?Goldantifadereducesphotobleaching抗淬滅封片劑選擇指南-幾乎不影響或很少影響熒光強(qiáng)度，優(yōu)越的抗淬滅效果即用型–滴瓶設(shè)計(jì)室溫保存和使用????含DAPI和不含DAPI兩種配方:適用于核復(fù)染LifeTechnologies?Proprietary|91ProLong?GoldAntifadeReagentSlowFade?GoldAntifadeReagentLong-termstorageYesYesYesYesNoNoNoNoImmediateviewingNoNoNo抗淬滅封片劑選擇指南-幾乎不影響或很少影響熒光強(qiáng)度，優(yōu)越的抗淬滅效果即用型–滴瓶設(shè)計(jì)室溫保存和使用????含DAPI和不含DAPI兩種配方:適用于核復(fù)染LifeTechnologies?Proprietary|91ProLong?GoldAntifadeReagentSlowFade?GoldAntifadeReagentLong-termstorageYesYesYesYesNoNoNoNoImmediateviewingNoNoNoNoYesYesYesYesCounterstainNoNoDAPIDAPINoNoDAPIDAPIFormat5x2mL5x2mLCat.No.P36934P36930P36935P36931S36937S36936S36939S36938SlowFade?Gold無需可立即觀察適用于快速評(píng)估ProLong?Gold需要適用于樣本的長期保存適用于 圖片的免疫熒光&一抗標(biāo)記染核封片二抗標(biāo)記Image-iT?固定/透化試劑盒DAPISYTOXImage-iTFX信號(hào)增強(qiáng)劑ProlongGoldSlowFade免疫熒光&一抗標(biāo)記染核封片二抗標(biāo)記Image-iT?固定/透化試劑盒DAPISYTOXImage-iTFX信號(hào)增強(qiáng)劑ProlongGoldSlowFadeGold一抗二抗1x即用可固定、透化并洗滌細(xì)胞；室溫保存；AlexaFluorDyes更亮、更穩(wěn)定用量低；多種熒光染料可供選擇降低背景熒光提高檢測(cè)靈敏度使用方便多種顏色可供選擇高品質(zhì)免單抗使用方便LifeTechnologies?Proprietary|92Agenda2.熒光檢測(cè)中常見問題和解決方案4.熒光標(biāo)記中的新“”和應(yīng)用LifeAgenda2.熒光檢測(cè)中常見問題和解決方案4.熒光標(biāo)記中的新“”和應(yīng)用LifeTechnologies?Proprietary|93GrowingApplicationsofGrowingApplicationsof―ClickChemistry‖(點(diǎn)擊化學(xué))一種反應(yīng)，無限可能LifeTechnologies?Proprietary|94ProteinLabelingChemistry:AProteinLabelingChemistry:ABriefOverview?CommonProteinLabelingTargetsAmine–Thiol–LifeTechnologies?Proprietary|95Amine–/thiol-reactivelabelingchemistriesPurifiedproteinsAmine–/thiol-reactivelabelingchemistriesPurifiedproteinsorotherbiopolymersNonselectiveinsitulabelingoftotalcellularthioloraminecontentInsitulabelingofspecificmolecularpopulations—newlysynthesizedLifeTechnologies?Proprietary|96Clickchemistry什么是“Clikc-it”化學(xué)？Click-it化學(xué)：由BarrySharpless在2001年提出，介紹了將小產(chǎn)生物質(zhì)的化學(xué)反應(yīng)。銅催化的疊氮-炔環(huán)加成反應(yīng)連在一起而快速可靠地AB可以是蛋白、熒什么是“Clikc-it”化學(xué)？Click-it化學(xué)：由BarrySharpless在2001年提出，介紹了將小產(chǎn)生物質(zhì)的化學(xué)反應(yīng)。銅催化的疊氮-炔環(huán)加成反應(yīng)連在一起而快速可靠地AB可以是蛋白、熒光基團(tuán)、核酸、半抗原、小―界的樂高”O(jiān)R問題/困擾：體積大的標(biāo)記&檢測(cè)為生物學(xué)分析帶來解決方案Click-iT?分析：特異、高效、生物正交的小分子化學(xué)反應(yīng)，作為生物學(xué)分析的平臺(tái)LifeTechnologies?Proprietary|975-Bromo-2’-deoxyuridine(BrdU)BrdU:Antibody-baseddetectionof5-Bromo-2’-deoxyuridine(BrdU)BrdU:Antibody-baseddetectionofthenucleotideanalogbromo-deoxyuridine(BrdU)IncorporatedintoDNAduringsynthesisasathymidineanalogDetectedusingananti-BrdUantibodyDifferentiateswhereincellcycleIncorporatedintodsDNAmakesBrdUfairlyinaccessibletotheBrdUantibodyRequiresDNAdenaturationtoincreaseaccessibilityHardtocontrolreproduciblyCelltypeshavesignificantdifferencesinsensitivitytoDNAdenaturationDenaturationcandestroycellularandaffectscellmorphologyHuge!?5-Bromo-2’-deoxyuridineLifeTechnologies?Proprietary|98傳統(tǒng)的BrdU技術(shù)原理新生DNA時(shí)，BrdU摻傳統(tǒng)的BrdU技術(shù)原理新生DNA時(shí)，BrdU摻入其中當(dāng)活細(xì)胞中BrBrBrBrdU5-溴-2‘-脫氧尿苷)BrLifeTechnologies?Proprietary|99BrdU問題：大量基于抗體的檢測(cè)探針無法接近BrdU問題：大量基于抗體的檢測(cè)探針無法接近摻入BrdU的完整DNABrBrBrLifeTechnologies?Proprietary|100BrBrdU在固定和透化之后，DNA必須經(jīng)過劇烈的樣BrdU在固定和透化之后，DNA必須經(jīng)過劇烈的樣品處理變性，讓抗體接近BrdUBrBrBrBrLifeTechnologies?Proprietary|101BrdUBrBrBrBrLifeTechnologies?BrdUBrBrBrBrLifeTechnologies?Proprietary|102BrdU技術(shù)特點(diǎn)及不足BrBr無放射性兼容多重分析，但需要分離雙鏈BrdU技術(shù)特點(diǎn)及不足BrBr無放射性兼容多重分析，但需要分離雙鏈，以便BrdU抗體接近，可能影響：其他抗體的結(jié)合能力形態(tài)Br需要雙鏈DNA才能高效結(jié)合的如細(xì)胞周期的能力，BrLifeTechnologies?Proprietary|103解決方案Click-iT?EdU新生解決方案Click-iT?EdU新生DNA時(shí)，EdU摻入其中當(dāng)活細(xì)胞中EdU5-乙炔基-2‘-脫氧尿苷)LifeTechnologies?Proprietary|104EdU在固定和透化之后，EdU可通EdU在固定和透化之后，EdU可通過銅催化的點(diǎn)擊反應(yīng)被疊氮報(bào)告基團(tuán)檢測(cè)LifeTechnologies?Proprietary|105解決方案Click-iT?EdU非放射性無需DNA變解決方案Click-iT?EdU非放射性無需DNA變性簡單的操作步驟小檢測(cè)兼容多重分析，包括其他抗體細(xì)胞周期分析的LifeTechnologies?Proprietary|106解決方案Click-iT?EdU解決方案Click-iT?EdU保留了細(xì)胞形態(tài)麂皮膚成纖維細(xì)胞用Click-iT?EdUAlexaFluor?647檢測(cè)(紫色)，以及微管（）、高爾基體（綠色）和過氧化物酶體（橙色）的免疫檢測(cè)。LifeTechnologies?Proprietary|107解決方案Click-iT?EdU適用于組織解決方案Click-iT?EdU適用于組織切片在Click-iT?操作之后，組織切片得到了很好的保留Click-iT?EdU操作標(biāo)準(zhǔn)的BrdU操作小鼠腸道切片：Hoechst33342（綠色）、AlexaFluor?594標(biāo)記的EdUBrdU檢測(cè)（紅色）Mildfixation&permeabilizationMaintaincell&tissuemorphologyMaintaindsDNAintegrityMaintainantigenrecognitionsitesLifeTechnologies?Proprietary|108HarshHClorDNasetreatmentrequiredLossofcell&tissuemorphologyLossofdsDNAintegrityLossofantigenrecognitionsitesFluor?594Hoechst33342*EdUAlexaFluor?594Hoechst33342*流程比較：Click-iT?EdUvs.BrdULifeTechnologies?Proprietary|109通過點(diǎn)擊化學(xué)測(cè)定細(xì)胞或組織中的增殖完成時(shí)間<2hours通過熒光顯微鏡、流式細(xì)流程比較：Click-iT?EdUvs.BrdULifeTechnologies?Proprietary|109通過點(diǎn)擊化學(xué)測(cè)定細(xì)胞或組織中的增殖完成時(shí)間<2hours通過熒光顯微鏡、流式細(xì)胞儀或高通量成像（HCS）來檢測(cè)BrdU的操作步驟Wash3X15minutesHClDenaturation40minutesNeutralize12minutesWash3X15minutesBlock60minutesAnti-BrdUincubation1-16hoursWash3X15minutesSecondaryantibodyincubation120minutesWash3X15minutesNuclearcounterstain15minutesWash3X15minutesImageTOTALTIME6-21hours點(diǎn)擊化學(xué)EdU的操作步驟Click-iT?detectionreaction30minutesWash2X10minutesNuclearcounterstain15minutesWash3X15minutesImageTOTALTIME70minutes用EdU或BrdU孵育，固定&透化樣品Click-iT?PlusEdUJustReleased!ProliferationClick-iT?PlusEdUJustReleased!ProliferationKits–DNASynthesisForInternalUseOnly.LifeTechnologies?Proprietary|110EdUGFPHoechstClick-iT?EdUversusClick-iT?PlusComparisonLifeTechnologies?Proprietary|111ClassicClick-iT?EdUClick-iT?PlusEdUSignalintensityBrightAsbrightorbrighterReactionrateFasterClick-iT?EdUversusClick-iT?PlusComparisonLifeTechnologies?Proprietary|111ClassicClick-iT?EdUClick-iT?PlusEdUSignalintensityBrightAsbrightorbrighterReactionrateFasterthanBrdUAsfastorfasterthanClassicClick-iT?EdUWorkflow~3hours(2hrEdUincubation,1hrClick-iT?Reaction)<3hours(<2hrEdUincubation,1hrClick-iT?Reaction)Q-dotcompatibleNoNoGFPcompatibleNoYESPhalloidincompatibleNoNomCherryYES–invitro;No–invivoYESRNAcompatibleNoNoR-PEcompatiblePost-Click-iT?reactionstainingisrequiredYESR-PEtandemcompatiblePost-Click-iT?reactionstainingisrequiredYESAPCcompatibleYESYESPerCPcompatibleYESYESBrilliantVioletdyesYESYESExample-Click-iT?PlusEdUProliferationClassicExample-Click-iT?PlusEdUProliferationClassicClick-iT?EdUAlexaFluor?594Click-iT?PlusEdUAlexaFluor?594ClassicClick-iT?EdUAlexaFluor?647Click-iT?PlusEdUAlexaFluor?647LifeTechnologies?Proprietary|112GFPfluorescencemaintainedGFPfluorescencemaintainedUsingtheUsingtheClick-iT?PlusAlexaFluor?555ImagingKittodetectinvivoproliferationandGFPsignalAnotherexampleoftheuseoftheClick-iT?PlusEdUforinvivodetectionofproliferationandretentionofthefluorescentsignalfromGFP.LifeTechnologies?Proprietary|113Click-iT?BasedDetectionofNascentClick-iT?BasedDetectionofNascentRNASynthesis“EU”containsanalkynemoietyNIH3T3cellsincubatedwith1mMEUfor1hrfollowedbyclickreactionwithAlexaFluor?647azide(magenta),immuno-detectionoftubulinwithAlexaFluor?488secondary(green),andHoechstnuclearcounterstain(blue).LifeTechnologies?Proprietary|114qRT-PCRiswidelyusedtotostudymRNAtranscriptionBut…WhatqRT-PCRiswidelyusedtotostudymRNAtranscriptionBut…Whatreallyhappened?LifeTechnologies?Proprietary|115Click-iTTechnology-OneReaction,EndlessClick-iTTechnology-OneReaction,EndlessPossibilities!Atransformingdiscoverytoolforsystemsbiologyresearch!LifeTechnologies?Proprietary|116A=AlkyneModifiedBiomoleculeB=AzidoModifiedDyeorconverseGeneExpression-NewRNASynthesisMacromoleculeEnrichment-ProteinEnrichmentCellDeath-DNAFragmentationCellGrowth&Differentiation-Post-TranslationalModificationsCellProliferation-NewDNASynthesisCellGrowth&Differentiation-NewProteinSynthesis物種（和界）的Click-iT?EdUClick-iT?EdU對(duì)小鼠嗅覺上皮細(xì)胞中出生日期差異細(xì)胞的標(biāo)記。F.Chehrehasaetal.物種（和界）的Click-iT?EdUClick-iT?EdU對(duì)小鼠嗅覺上皮細(xì)胞中出生日期差異細(xì)胞的標(biāo)記。F.Chehrehasaetal.JNeurosciMethod(2009)用Click-IT?EdU標(biāo)記雞胚胎中的神經(jīng)管和聽泡。M.Warrenetal.Dev.Dynamics(2009)用Click-iT?EdU檢測(cè)線蟲中生殖細(xì)胞的增殖。M.Dorsettetal.Genetics(2009)以Click-iT?EdU標(biāo)記的紫花苜蓿懸浮培養(yǎng)物。ImagecourtesyofFerhanAyadin,CellularImagingLaboratory,BiologicalResearchCenter海洋扁形蟲courtesyofJulianP.S.SmithIII,WinthropMicroscopyFacility,大腸桿菌斑馬魚幼蟲ImagecourtesyofSarahCheesman,UniversityofOregon裂殖酵母Methods(2009)48:8-13WinthropUniversityImagecourtesyofSarahA.Sabatinos,UniversityofSouthernCaliforniaLifeTechnologies?Proprietary|117MolecularProbes-熒光標(biāo)記和檢測(cè)的?實(shí)現(xiàn)細(xì)胞分析的工具30年來引領(lǐng)熒光技術(shù)30,000篇物?生命科學(xué)中被和化學(xué)廣泛的熒光?提供可見光譜中幾乎任一種顏色旗艦Fluor?是行業(yè)標(biāo)準(zhǔn)（如MolecularProbes-熒光標(biāo)記和檢測(cè)的?實(shí)現(xiàn)細(xì)胞分析的工具30年來引領(lǐng)熒光技術(shù)30,000篇物?生命科學(xué)中被和化學(xué)廣泛的熒光?提供可見光譜中幾乎任一種顏色旗艦Fluor?是行業(yè)標(biāo)準(zhǔn)（如Alexa）LifeTechnologies?Proprietary|118MolecularProbesMolecularProbes?細(xì)胞結(jié)構(gòu)LifeTechnologies?Proprietary|119講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?Proprietary|講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?Proprietary|細(xì)胞的形狀和結(jié)構(gòu)能告訴我們什么功能潛在的疾病發(fā)展?fàn)顩r高度動(dòng)態(tài)的信息非常適像LifeTechnologies?Proprietary細(xì)胞的形狀和結(jié)構(gòu)能告訴我們什么功能潛在的疾病發(fā)展?fàn)顩r高度動(dòng)態(tài)的信息非常適像LifeTechnologies?Proprietary|122細(xì)胞包含了保守的細(xì)胞內(nèi)結(jié)構(gòu)Life細(xì)胞包含了保守的細(xì)胞內(nèi)結(jié)構(gòu)LifeTechnologies?Proprietary|123各種各樣的熒光探針，適用于活細(xì)胞和固定細(xì)胞中的結(jié)構(gòu)WebTool:各種各樣的熒光探針，適用于活細(xì)胞和固定細(xì)胞中的結(jié)構(gòu)WebTool:OrganelleStainsSelectionGuideforFluorescenceImagingLifeTecholoiepitry4構(gòu)海報(bào)講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?Proprietary|各種各樣的熒光探針，適用于活細(xì)胞和固定細(xì)胞抗體編碼的熒光結(jié)合物??蛋白熒光和毒素、、物瞬時(shí)表達(dá)測(cè)導(dǎo)入實(shí)現(xiàn)?異靶點(diǎn)固定細(xì)胞標(biāo)記結(jié)合性質(zhì)或特?活細(xì)胞標(biāo)記但可固定?活細(xì)胞和固定細(xì)胞標(biāo)記LifeTechnologies?Proprietary|各種各樣的熒光探針，適用于活細(xì)胞和固定細(xì)胞抗體編碼的熒光結(jié)合物??蛋白熒光和毒素、、物瞬時(shí)表達(dá)測(cè)導(dǎo)入實(shí)現(xiàn)?異靶點(diǎn)固定細(xì)胞標(biāo)記結(jié)合性質(zhì)或特?活細(xì)胞標(biāo)記但可固定?活細(xì)胞和固定細(xì)胞標(biāo)記LifeTechnologies?Proprietary|126細(xì)胞器選擇性的各種各樣的熒光，可根據(jù)其化學(xué)性質(zhì)對(duì)活細(xì)胞中的細(xì)胞結(jié)構(gòu)進(jìn)行染色……MitoTracker?，適用于線粒體核酸，適用于細(xì)胞核和核仁，適用于酸性區(qū)室LysoTracker?&LysoSensor?親脂性ER-Tracker細(xì)胞器選擇性的各種各樣的熒光，可根據(jù)其化學(xué)性質(zhì)對(duì)活細(xì)胞中的細(xì)胞結(jié)構(gòu)進(jìn)行染色……MitoTracker?，適用于線粒體核酸，適用于細(xì)胞核和核仁，適用于酸性區(qū)室LysoTracker?&LysoSensor?親脂性ER-Tracker，適用于膜，適用于內(nèi)質(zhì)網(wǎng)神經(jīng)酰胺結(jié)合物，適用于高爾基體龐大的工具箱，適合短期的活細(xì)胞成像實(shí)驗(yàn)（一些可固定）LifeTechnologies?Proprietary|127熒光蛋白：依性質(zhì)設(shè)計(jì)的奇妙工具，但是問題：???傳統(tǒng)的細(xì)胞導(dǎo)入不夠高效，還可能需要專業(yè)的生物學(xué)知識(shí)過程漫長，流程復(fù)雜...直到現(xiàn)在?解決方案：熒光蛋白：依性質(zhì)設(shè)計(jì)的奇妙工具，但是問題：???傳統(tǒng)的細(xì)胞導(dǎo)入不夠高效，還可能需要專業(yè)的生物學(xué)知識(shí)過程漫長，流程復(fù)雜...直到現(xiàn)在?解決方案：適用于活細(xì)胞的熒光蛋白，通過BacMam導(dǎo)入可進(jìn)入一切細(xì)胞CellLight?&Premo?傳感器LifeTechnologies?Proprietary|128講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?講座大綱為什么細(xì)胞結(jié)構(gòu)在細(xì)胞生物學(xué)中如此重要？探索細(xì)胞結(jié)構(gòu)的方法：、抗體&熒光蛋白細(xì)胞結(jié)構(gòu)的觀察變得簡單：BacMam導(dǎo)入MolecularProbes?的新的解決方案簡化活細(xì)胞成像Technologies?Proprietary|導(dǎo)入技術(shù)BacMam是帶有哺乳動(dòng)物啟動(dòng)子的昆蟲桿狀BacMam?用于將效率?高效轉(zhuǎn)導(dǎo)對(duì)細(xì)胞幾乎無毒性規(guī)?？勺兒唵畏奖阒恍鑼踩尤爰?xì)胞在哺乳動(dòng)物細(xì)胞內(nèi)BSL1生物安全防護(hù)水平轉(zhuǎn)導(dǎo)難以轉(zhuǎn)染的細(xì)胞導(dǎo)入技術(shù)BacMam是帶有哺乳動(dòng)物啟動(dòng)子的昆蟲桿狀BacMam?用于將效率?高效轉(zhuǎn)導(dǎo)對(duì)細(xì)胞幾乎無毒性規(guī)?？勺兒唵畏奖阒恍鑼踩尤爰?xì)胞在哺乳動(dòng)物細(xì)胞內(nèi)BSL1生物安全防護(hù)水平轉(zhuǎn)導(dǎo)難以轉(zhuǎn)染的細(xì)胞原代細(xì)胞干細(xì)胞神經(jīng)元BacMam是一種高效，方便并且安全的LifeTechnologies?Proprietary|130導(dǎo)入技術(shù)簡單的流程CellLight?ER-GFPGeorgePatterson,簡單的流程CellLight?ER-GFPGeorgePatterson,Lippincott-Schwartz,NIH實(shí)現(xiàn)了更長時(shí)間、更高內(nèi)涵的CellLight?Mito-GFPMichaelDavidson,FloridaStateUniversity活細(xì)胞成像LifeTechnologies?Proprietary|131LifeLifeTechnologies?Proprietary|132Live-cellimagingLive-cellimagingwithCellLight?EarlyEndosomes-RFPandMitochondria-GFPinHeLacells.Live-cellimagingwithCellLight?EarlyEndosomes-RFPandMitochondria-GFPinHeLacells.HeLacellsweretransducedwithCellLight?Endosomes-RFP(Cat.No.C10587)andMitochondria-GFP(Cat.No.C10600),thenandco-stainedHoechst33342(Cat.No.H24192).ImagingwasperformedonlivecellsusingaDeltaVision?CoremicroscopeandstandardDAPI/FITC/TRITCfiltersets.LifeTechnologies?Proprietary|133LivecellLivecellimagingwithCellLight?reagents.HeLacellsweretransducedwithCellLight?Mitochondria-GFP(Cat.No.C10600)and(Cat.no.C10612).Thefollowingday,cellswerestainedwithHoechst33342(Cat.No.H24192)andimagingwasperformedonlivecellsusingaDeltaVision?CoremicroscopeandstandardDAPI/FITC/TRITCfiltersets.LifeTechnologies?Proprietary|134CellLight?試劑實(shí)現(xiàn)了細(xì)胞結(jié)構(gòu)的更長時(shí)間活細(xì)胞成像問題：熒光的長時(shí)間活細(xì)胞成像會(huì)導(dǎo)致光毒性，并伴隨著光漂白15分鐘內(nèi)每30秒CellLight?試劑實(shí)現(xiàn)了細(xì)胞結(jié)構(gòu)的更長時(shí)間活細(xì)胞成像問題：熒光的長時(shí)間活細(xì)胞成像會(huì)導(dǎo)致光毒性，并伴隨著光漂白15分鐘內(nèi)每30秒獲取圖像一次Actin-RFPPM-GFPActin-RFP解決方案示例CellLigh