流式细胞仪原理及应用

流式細胞儀---FACSCalibur之基本原理與應用產品專員劉益年美商必帝公司大綱實驗設計原理流式細胞儀運作原理流式細胞儀之應用IntroductionoftheexperimentdesignforFlowCytometry藉由螢光抗體標識細胞之抗原特性藉由螢光化合物標識細胞特性藉由螢光染劑標識細胞特性藉由螢光蛋白標識細胞特性CytometryBeadsArray單一細胞特性之偵測單一細胞特性之偵測AdhesionReceptorsMetaboliccytokinesstructureenzymesLysedWholeBloodComponentsGranulocytesMonocytesEosinophilsBasophilsNeutrophilsLymphocytesTBNKTHelperTCytotoxicPeripheralWhiteBloodCellsCD45+CD3+CD4+CD3+CD8+CD3+CD3-CD19+HLA-DR+CD3-CD16+CD56+MonocytesExample訊息傳導BD™PhosFlowforWholeBloodorPBMCSamples實驗設計原理藉由螢光抗體標識細胞之抗原特性藉由螢光化合物標識細胞特性藉由螢光染劑標識細胞特性藉由螢光蛋白標識細胞特性CytometryBeadsArrayAnnexinVAssayFlowCytometricAnalysisofApoptosisinJurkatTCellsRelativeCellNumberAnnexinV–PE00.5124IncubationwithCamptothecin(hr)Fas-inducedApoptosisinJurkatCellsPIAnnexinV-FITC實驗設計原理藉由螢光抗體標識細胞之抗原特性藉由螢光化合物標識細胞特性藉由螢光染劑標識細胞特性藉由螢光蛋白標識細胞特性CytometryBeadsArrayDNA特異性染劑N+C2H5NH2NH2EthidiumN+C2H2)3NH2NH2N+C2H5CH3C2H5PropidiumG2MG0G1s02004006008001000G0G1sG2MDNAcontentCount2N4N細胞周期位相的決定SubG1細胞增殖反應細胞增殖反應實驗設計原理藉由螢光抗體標識細胞之抗原特性藉由螢光化合物標識細胞特性藉由螢光染劑標識細胞特性藉由螢光蛋白標識細胞特性CytometryBeadsArrayGFPasReporterGFPasaReporterGFPasaReporterTheUsageofGFPGervaix,A.et.al.1997.AnewreportercelllinetomonitorHIVinfectionanddrugsusceptibilityinvitro.PNASvol.94.GFPasaReporterGFPasaReporter實驗設計原理藉由螢光抗體標識細胞之抗原特性藉由螢光化合物標識細胞特性藉由螢光染劑標識細胞特性藉由螢光蛋白標識細胞特性CytometryBeadsArrayCytometricBeadsArray(CBA)SingleStepIncubationTwo-StepIncubationorBeadsProvideaFlexiblePlatformMultiplesizesBeadsprovideanexpandableassayplatformforusewithaflowcytometerDifferentintensities*DifferentcolorswithdifferentintensitiesProteinsMeasuredA.Interleukin(IL)-2B.IL-4C.IL-5D.IL-10E.TumorNecrosisFactor-aF.Interferon-gZap70Detection.KineticanalysisofTcellactivationbyanti-CD3/CD28PhiladelphiachromosomeThepresenceofthistranslocationisahighlysensitivetestforCML,since95%ofpeoplewithCMLhavethisabnormalityPhiladelphiachromosome流式細胞儀---FACSCalibur運作原理緩衝液液流區緩衝液液流區樣品流動區偵測區激發光源螢光散射光流式細胞儀的工作原理流式細胞儀能測量:散射光細胞大小(前方散射光)細胞折射率(側方散射光)各色螢光液流系統:將細胞依序送到測量區受檢。光學系統:產生並收集螢光、光散射等信號。電子系統:將光學訊號轉換成電子訊號。 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 所輸出的電流訊號，以脈衝高度、寬度、積分面積顯示。量化訊號並傳至電腦。綜合三個系統的功能:綜合三個系統的功能-液流系統FACSCalibur的液流系統FACSCalibur儀 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 板LO=12uL/minMED=35uL/minHI=60uL/minPRIME=Drain&FillSTANDBYRUN緩衝液液流區緩衝液液流區樣品流動區偵測區激發光源螢光散射光FACSCalibur的工作原理綜合三個系統的功能-光學系統螢光濾片FACSCalibur的光學系統488nm綜合三個系統的功能-電子系統將光學訊號轉換成正比例的電子訊號。分析所輸出的電子訊號，以脈衝高度、寬度、積分面積顯示。將量化訊號傳至電腦。電子系統電子系統電位脈衝之定量訊號之產生與處理AmpGain1.0-9.99TheUseofLinearAmplificatonAssumeweconvertlinearanalogsignalsusinga10bitADC--wehave1024channelsofrangecorrespondingto0-10V.Channeldifferenceis10V/1024=0.01VIdealLinToLogConversion實驗數據的呈現15to20mmMonocyte10to14mmNeutrophil8to10mmLymphocyte散點圖反映細胞形態常見的數據呈現直方圖分析報告CV=S.D./Mean散點圖分析報告FACSCalibur-複習綜合三個系統的功能流式細胞技術於醫事技術上之應用臨床微生物檢測工具之研發監測疾病病程與治療預後狀況敗血症之研發輸血醫學相關研究抗藥性研究感染症相關研究免疫功能相關研究凝血疾病相關研究DNA分析與腫瘤診斷定量分析工具之研發血腫疾病研究癌症相關研究流式細胞技術於生物技術開發之應用單珠抗體研發與螢光標識抗體之生產癌症診斷試劑研發老化研究〈細胞凋亡、自由基等〉免疫功能與相關疾病診斷，治療與監控工具之開發細胞生物學研究﹝細胞毒性，氧化代謝，訊息傳導等﹞商用檢測試劑組開發新藥開發造血幹細胞分化研究遺傳探針之研發病原特定性T細胞偵測工具之開發細菌基因鑑定轉染工具-報導基因之螢光蛋白ApoptoticPhenotypesMorphologicaldisintegration•Plasmamembranelosesasymmetry(1-2h)•DNAfragmentation(6-8hbyTUNEL,12-24hbyHypoploidyAnalysis)•Cellshrinkage(12-24h)•Chromatincondensation(18-36hbygelanalysis)•Cellsarephagocytosedbeforelossofmembraneintegrity-NoinflammatoryresponseApoptoticPhenotypesBiochemicalPhenotypicChanges•Calciuminflux,pHchanges(0.5-1h)•Energypumpshutsoff(0.5-1h)•Mitochondriadepolarization(1-3h)•Caspase-3activation(2-3h)FlowCytometryAssaysLive(unfixedcells)•AnnexinV-PI分析法•MitoScreen(JC-1)Fixedcells•ActiveCaspase-3•APO-BRDU™(DNA鏈斷分析法)•HypoploidyAnalysis(Sub-G1分析法)AnnexinVAssayAnnexinVisaQuickAssayApoptoticandControlCellsWashwithPBSTwocolorOnecolorPropidiumiodideandLabeledAnnexinVAnalyzebyFlowCytometryIncubate15minLabeledAnnexinVAnnexinV-PIAnalysisR4R1R2R3PIAnnexin-VFITCAFSCSSCgatedR1BFSCSSCgatedR3CFSCSSCgatedR2DANALYSISOFVIABLEAPOPTOTICCELLSAnnexinVAssay:ATimeCoursePIAnnexinV-FITCFlowCytometryAssaysLive(unfixedcells)•AnnexinV-PI分析法•MitoScreen(JC-1)Fixedcells•ActiveCaspase-3•APO-BRDU™(DNA鏈斷分析法)•HypoploidyAnalysis(Sub-G1分析法)Mitoscreen(JC-1):MonitorMitochondrialMembranePotential(Dy)•EnergystoredasanegativeelectrochemicalgradientDypolarized•ApoptosisisoftenassociatedwithadecreaseinDyDydepolarizedJC-1:•1stJ-aggregate-formingCationicdyefoundtobeDysensitive•FluorescencecolordependentonDyAnalysisbyflowcytometry:•Dypolarized(J-aggregates):FITC-PEchannel•Dydepolarized(monomers):FITCchannelModelSystem:MouseApoptosisCD4-PerCP+CD8-APCMouseThymocytesControl(3hr)JC-1FasmAb+ProteinG(3hr)FlowCytometryJC-1StaininginMouseThymocytesControl(3hr)FasmAb(3hr)ControlFasmAbJC-1StaininginMouseThymocyteSubpopulationsJC-1(FL-1)R6R5R4R3JC-1(FL-2)CD8-APCCD4-PerCPGatedonCD4/CD8FlowCytometryAssaysLive(unfixedcells)•AnnexinV-PI分析法•MitoScreen(JC-1)Fixedcells•ActiveCaspase-3•APO-BRDU™(DNA鏈斷分析法)•HypoploidyAnalysis(Sub-G1分析法)FADDPro-caspase–8ReceptorMitochondrialActivecaspase–8?StaurosporineCamptothecinDNAdamageActiveBid+Pro-caspase–9+Apaf-1+CytcActivecaspase–9Activecaspase–3(6,7)MassivesubstratecleavageZ–VADZ–VADMitochondriaApoptosisSignalingFasPro-caspase–3(6,7)Z–VADDEATHFasLApoptosisValidationCytofix/Cytoperm™JurkatTCellsIncubationincamptothecin0,0.5,1,2or4hrAnnexinV–PEFlowCytomertyAnnexinVandCaspase-3StainingProtocolActiveCaspase-3–FITCFlowCytometryFlowCytometricAnalysisofApoptosisinJurkatTCellsRelativeCellNumberActiveCaspase-3–FITCAnnexinV–PE00.5124IncubationwithCamptothecin(hr)FlowCytometryAssaysLive(unfixedcells)•AnnexinV-PI分析法•MitoScreen(JC-1)Fixedcells•ActiveCaspase-3•APO-BRDU™(DNA鏈斷分析法)•HypoploidyAnalysis(Sub-G1分析法)APO-BRDU™ExploitstheLargeNumberofDNAFragmentsinApoptoticCells•DNA3’-hydroxylterminalarelabelledwithbromolateddeoxyuridinetriphosphatenucleotides(Br-dUTP)-Reactioncatalyzedbyterminaldeoxynucleodidyltransferase(TdT)•Non-apoptoticcellslackexposed3’-hydroxylendsandthereforeincorporatelittleBr-dUTP•Br-dUTPsitesaredetectedwithaBrdU-FITCmAbAPO-BRDU™AssayGreenFluorescenceAPO-BRDU™AssayDNAContentSideScatterForwardScatterR2:ApoptoticCellsR1:NormalCellsGreenFLAPO-BRDU™AssayFas-inducedApoptosisinJurkatCellsPIAnnexinV-FITCBrdU-FITCPIFlowCytometryAssaysLive(unfixedcells)•AnnexinV-PI分析法•MitoScreen(JC-1)Fixedcells•ActiveCaspase-3•APO-BRDU™(DNA鏈斷分析法)•HypoploidyAnalysis(Sub-G1分析法)染色原則同細胞周期分析OvernightincubationtoextractsmallDNAfragments,thenPIstain.Pre-treatwithTritonX.Useofphosphatebufferswithhighosmolarity.Sub-G1分析法細胞周期與DNA含量DNA直方圖-理論與實際G2MG0G1s02004006008001000G0G1sG2MDNAcontentCount2N4N細胞周期位相的決定典型的DNA直方圖G0-G1SG2-MFluorescenceIntensity#ofEvents檢體前處理的注意事項Healthy,viablecellsbeforeethanolfixation.Foradherentcells,avoidover-trypsinization.Useserumcontainingmediumtostoptrypsinization.Addethanolwhilevortexing.AvoidthereuseofRNase.Sub-G1分析法PIFluorescenceIntensity#ofEventsApoptoticcellsSub-G1分析法Sub-G1分析法FlowCytometryAssaysLive(unfixedcells)•AnnexinV-PI分析法•MitoScreen(JC-1)Fixedcells•ActiveCaspase-3•APO-BRDU™(DNA鏈斷分析法)•HypoploidyAnalysis(Sub-G1分析法)ApoptoticSampleCellsCellExtractsTissueSectionsFlowMicroscopyFlowELISAWesternBlot/ImmunoprecipitationSpectrofluorometryGeneExpressionIHCApoptosisDecisionTree謝謝！