流式细胞(FACS)多色组合原则和工具及新染料介绍ppt课件

多色组合原则和工具新染料介绍,李彩aixia_li,多色组合原则,按照机器设置染料的亮度与抗原表达相匹配同种细胞的Marker染料重叠最小化尽量避免联合使用带来的假阳性如果可能，用红激光做自发荧光高的样本。,染料选择-按照机器设置,1,表达强弱-AntigenDensity,染料选择,2,荧光染料的强度,CD5,CD8,染料选择,2,Example,高表达的抗体可用不太亮的染料，低/弱表达的Marker用亮的染料Example:CD8“bright”PacificBlueCD7“lessbright”PECD8=90Kmolecules/cellCD7=20Kmolecules/cell,2,.,7,EightcolorantibodypanelsproposedbytheHumanImmunophenotypingConsortium,NatRevImmunol,2012,.,8,Theimportanceofantibodychoice,ThestainingpatternsoftwocommerciallyavailableclonesofhumanCD38specificantibodyareverydifferent,despitethefactthatbothantibodieswereconjugatedtoallophycocyanin(APC)bythesamevendor,andwereusedtostainperipheralbloodmononuclearcells(PBMCs)fromthesamehealthysubjectunderidenticalconditions.V450,violet450.DatacourtesyofAngeliqueBiancotto,NationalHeart,LungandBloodInstitute,USA.,NatRevImmunol,2012,荧光染料光谱重叠最小化,spectraviewer,3,光谱重叠会导致数据丢失,CD45-FITCDimCD4-PE,CD45FITC漏到PE，CD4PE弱信号分不开,CD45-PerCPDimCD4-PE,CD45PerCP不漏到PE，弱CD4可以分开,3,Uncompensated,Compensated,dataspreadduetospillover,采用多激光激发减少重叠,同一细胞的抗原,用不同激光激发减少重叠,Example:CD3“bright”APC-Cy7CD7“lessbright”PEBothantigensexpressedonsamecell,lowspilloverofCD3intoCD7andviceversa.CD3=124Kmolecules/cellCD7=20Kmolecules/cell,3,双激发荧光染料,荧光染料可被不止1laser激发，导致光谱重叠干扰，影响结果.AmCyan可被Violet（405nm)和Blue（488nm)(FITCdetector).PE-Cy5可漏到APCdetector.,WithoutCD45AmCyan:,WithCD45AmCyan:,CD19FITC,Onlyanissuewhenthetwomarkers(CD45andCD19)areco-expressedonthesamecellpopulation.,3,荧光染料选择,避免染料和其衍生物（tandem-dye）在同一细胞上标记，或者选用更稳定的tandem-dye,4,不同细胞,不同细胞,由于tandem-dye降解会产生加阳性,A.,FalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7,FalsepositivesinPEchannelremain,CD8APC-Cy7+cells,CD4PE-Cy7+cells,B.,WithCD8APC-Cy7andCD4PE-Cy7,WithoutCD8APC-Cy7,4,补偿:Tandems,0hours,2hours,22.5hours,PE,CD8,CD3,PE-Cy5,PE-Cy7,样本曝光时间,4,PerCP,NewTandemsAreMoreStable,APC-H7toreplaceAPC-Cy7:,ComparisonofSampleStability,(inBDStabilizingFixativeatRT),0,50,100,150,200,250,0,1,2,4,6,8,24,48,Hoursoflightexposure,%Spillover,4,自发荧光多的选择用红激光,染料选择,Perfect!,5,.,18,Identificationofimmunecellsubsetsbyeightcolourantibodystaining,NatRevImmunol,2012,.,19,Identificationofimmunecellsubsetsbyeightcolourantibodystaining,NatRevImmunol,2012,.,20,Eight-colorforhematopoieticstemcell,MPPs:multipotentprogenitorcellsCMPs:commonmyeloidprogenitorcellsCLPs:commonlymphoidprogenitorcellsMEPs:megakaryocyteerythroidprogenitorcellsGMPs:granulocytemacrophageprogenitorcells,.,21,Eight-colorforhematopoieticstemcell,.,22,Eight-colorforhematopoieticstemcell,.,23,Six-colorforhematopoieticstemcell,Buffer选择,FixationbufferBDCytofix/CytopermandBDPerm/WashbufferBDPharmingenFoxP3bufferset(mouseorhuman)BDPhosflowPermBufferIIBDPhosflowPermBufferIIIBDIntraSurekit,EffectofBDCytofix/CytopermBufferonMouseFoxp3Staining,MouseFoxp3AlexaFluor647,Foxp3Buffer,CD4PE,BDCytofix/Cytoperm,背景处理,TheImmunoglobulin,背景处理-Blocking,FcBlockMouseFcBlock,purifiedCD16/32cat#553141/553142ReducesBackgroundStaining,Nopre-inc.FcBlockPre-inc.FcBlock,28,多色应用工具,29,1,30,2,31,3,32,33,34,35,36,37,38,39,40,41,42,43,新染料,44,PE-CF594,激发Laser：488nm或561nm发射波：612nm,更亮更稳定溢漏更少,FITCPEPE-CF594PerCPPE-CY7,45,更亮的PE-CF594,46,更稳定的PE-CF594,PE-CF594reagentshaveconsistentspillovervaluesbetweenlotsandspecificities,minimizingtheneedforlotspecificcompensationcontrols,BrilliantViolet421,48,BrilliantViolet421,ExMax:407nmEmMax:421nmand448nm用标准的HorizonV450filter:450/50nm,49,BrilliantViolet421：极亮,50,极亮：更好的细胞分群,亮的染料使细胞分群更明显阳性率增加,PerCP-Cy5.5,CD279,PE,BrilliantViolet421,20%,26%,31%,CD184,44%,79%,51,TregPanel12,CD127A647,CD25BV421,CD25PE,CD127BV421,CD127vsCD25,52,溢漏更少,BV421,V450,V500,53,稳定：StabilityinPFAfixatives,54,BV421特点总结,极亮溢漏更少稳定,55,应用：Multicolor,BV421是多色组合的理想选择尤其是弱表达/低表达Panels（FACSVerse）,很亮的10色组合,56,APC-H7,APC-H7,ComparisonofSampleStability,(inBDStabilizingFixativeatRT),0,50,100,150,200,250,0,1,2,4,6,8,24,48,Hoursoflightexposure,%Spillover,57,BDHorizonV450，BDHorizonV500,BDHorizonV450，BDHorizonV500,V450-PacificBlueV500-PacificOrangeAmCyan,Apoptosis,DNADamageandCellProliferationKit,59,theApoptosis,DNADamageandCellProliferationKit,同一管分析凋亡、DNA损伤和细胞增殖Apoptosis-cleavedPARP(PE)DNADamage-H2AX(AlexaFluor647)CellProliferationBrdU(PerCP-Cy5.5)省时、节约珍贵样本,60,SampleData,Withcamptothecintreatment(bottom)cellproliferationgoesdown(BrdU+cells),