流式細(xì)胞(FACS)多色組合原則和工具及新染料介紹 教育學(xué)習(xí)

多色組合原則和工具新染料介紹,李彩aixia_li,多色組合原則,按照機(jī)器設(shè)置染料的亮度與抗原表達(dá)相匹配同種細(xì)胞的Marker染料重疊最小化盡量避免聯(lián)合使用帶來的假陽性如果可能，用紅激光做自發(fā)熒光高的樣本。,染料選擇-按照機(jī)器設(shè)置,1,表達(dá)強(qiáng)弱-AntigenDensity,染料選擇,2,熒光染料的強(qiáng)度,CD5,CD8,染料選擇,2,Example,高表達(dá)的抗體可用不太亮的染料，低/弱表達(dá)的Marker用亮的染料Example:CD8“bright”PacificBlueCD7“l(fā)essbright”PECD8=90Kmolecules/cellCD7=20Kmolecules/cell,2,EightcolorantibodypanelsproposedbytheHumanImmunophenotypingConsortium,NatRevImmunol,2012,7,優(yōu)質(zhì)課件,Theimportanceofantibodychoice,ThestainingpatternsoftwocommerciallyavailableclonesofhumanCD38specificantibodyareverydifferent,despitethefactthatbothantibodieswereconjugatedtoallophycocyanin(APC)bythesamevendor,andwereusedtostainperipheralbloodmononuclearcells(PBMCs)fromthesamehealthysubjectunderidenticalconditions.V450,violet450.DatacourtesyofAngeliqueBiancotto,NationalHeart,LungandBloodInstitute,USA.,NatRevImmunol,2012,8,優(yōu)質(zhì)課件,熒光染料光譜重疊最小化,spectraviewer,3,光譜重疊會導(dǎo)致數(shù)據(jù)丟失,CD45-FITCDimCD4-PE,CD45FITC漏到PE，CD4PE弱信號分不開,CD45-PerCPDimCD4-PE,CD45PerCP不漏到PE，弱CD4可以分開,3,Uncompensated,Compensated,dataspreadduetospillover,采用多激光激發(fā)減少重疊,同一細(xì)胞的抗原,用不同激光激發(fā)減少重疊,Example:CD3“bright”APC-Cy7CD7“l(fā)essbright”PEBothantigensexpressedonsamecell,lowspilloverofCD3intoCD7andviceversa.CD3=124Kmolecules/cellCD7=20Kmolecules/cell,3,雙激發(fā)熒光染料,熒光染料可被不止1laser激發(fā)，導(dǎo)致光譜重疊干擾，影響結(jié)果.AmCyan可被Violet（405nm)和Blue（488nm)(FITCdetector).PE-Cy5可漏到APCdetector.,WithoutCD45AmCyan:,WithCD45AmCyan:,CD19FITC,Onlyanissuewhenthetwomarkers(CD45andCD19)areco-expressedonthesamecellpopulation.,3,熒光染料選擇,避免染料和其衍生物（tandem-dye）在同一細(xì)胞上標(biāo)記，或者選用更穩(wěn)定的tandem-dye,4,不同細(xì)胞,不同細(xì)胞,由于tandem-dye降解會產(chǎn)生加陽性,A.,FalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7,FalsepositivesinPEchannelremain,CD8APC-Cy7+cells,CD4PE-Cy7+cells,B.,WithCD8APC-Cy7andCD4PE-Cy7,WithoutCD8APC-Cy7,4,補(bǔ)償:Tandems,0hours,2hours,22.5hours,PE,CD8,CD3,PE-Cy5,PE-Cy7,樣本曝光時間,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,自發(fā)熒光多的選擇用紅激光,染料選擇,Perfect!,5,Identificationofimmunecellsubsetsbyeightcolourantibodystaining,NatRevImmunol,2012,18,優(yōu)質(zhì)課件,Identificationofimmunecellsubsetsbyeightcolourantibodystaining,NatRevImmunol,2012,19,優(yōu)質(zhì)課件,Eight-colorforhematopoieticstemcell,MPPs:multipotentprogenitorcellsCMPs:commonmyeloidprogenitorcellsCLPs:commonlymphoidprogenitorcellsMEPs:megakaryocyteerythroidprogenitorcellsGMPs:granulocytemacrophageprogenitorcells,20,優(yōu)質(zhì)課件,Eight-colorforhematopoieticstemcell,21,優(yōu)質(zhì)課件,Eight-colorforhematopoieticstemcell,22,優(yōu)質(zhì)課件,Six-colorforhematopoieticstemcell,23,優(yōu)質(zhì)課件,Buffer選擇,FixationbufferBDCytofix/CytopermandBDPerm/WashbufferBDPharmingenFoxP3bufferset(mouseorhuman)BDPhosflowPermBufferIIBDPhosflowPermBufferIIIBDIntraSurekit,EffectofBDCytofix/CytopermBufferonMouseFoxp3Staining,MouseFoxp3AlexaFluor647,Foxp3Buffer,CD4PE,BDCytofix/Cytoperm,背景處理,TheImmunoglobulin,背景處理-Blocking,FcBlockMouseFcBlock,purifiedCD16/32cat#553141/553142ReducesBackgroundStaining,Nopre-inc.FcBlockPre-inc.FcBlock,多色應(yīng)用工具,28,1,29,2,30,3,31,32,33,34,35,36,37,38,39,40,41,42,新染料,43,PE-CF594,激發(fā)Laser：488nm或561nm發(fā)射波：612nm,更亮更穩(wěn)定溢漏更少,FITCPEPE-CF594PerCPPE-CY7,44,更亮的PE-CF594,45,更穩(wěn)定的PE-CF594,PE-CF594reagentshaveconsistentspillovervaluesbetweenlotsandspecificities,minimizingtheneedforlotspecificcompensationcontrols,46,BrilliantViolet421,BrilliantViolet421,ExMax:407nmEmMax:421nmand448nm用標(biāo)準(zhǔn)的HorizonV450filter:450/50nm,48,BrilliantViolet421：極亮,49,極亮：更好的細(xì)胞分群,亮的染料使細(xì)胞分群更明顯陽性率增加,PerCP-Cy5.5,CD279,PE,BrilliantViolet421,20%,26%,31%,CD184,44%,79%,50,TregPanel12,CD127A647,CD25BV421,CD25PE,CD127BV421,CD127vsCD25,51,溢漏更少,BV421,V450,V500,52,穩(wěn)定：StabilityinPFAfixatives,53,BV421特點總結(jié),極亮溢漏更少穩(wěn)定,54,應(yīng)用：Multicolor,BV421是多色組合的理想選擇尤其是弱表達(dá)/低表達(dá)Panels（FACSVerse）,很亮的10色組合,55,APC-H7,APC-H7,ComparisonofSampleStability,(inBDStabilizingFixativeatRT),0,50,100,150,200,250,0,1,2,4,6,8,24,48,Hoursoflightexposure,%Spillover,56,BDHorizonV450，BDHorizonV500,BDHorizonV450，BDHorizonV500,V450-PacificBlueV500-PacificOrangeAmCyan,57,Apoptosis,DNADamageandCellProliferationKit,theApoptosis,DNADamageandCellProliferationKit,同一管分析凋亡、DNA損傷和細(xì)胞增殖Apoptosis-cleavedPARP(PE)DNADamage-H2AX(AlexaFluor647)CellProliferationBrdU(PerCP-Cy5.5)省時、節(jié)約珍貴樣本,59,SampleData,Withcamptothecintreatment(bottom)cellproliferationgoesdown(BrdU+cells),DN