流式细胞(FACS)多色组合原则和工具及新染料介绍

多色组合原则和工具新染料介绍李彩霞[e-mail]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6-color8-colorAdditionalFITCorAlexa488FITCorAlexa488FITCorAlexa488PEPEPEPE-CF594orPE-TexasRedorPE-Alexa610/594PE-CF594orPE-TexasRedorPE-Alexa610/594PerCP/PE-Cy5/PerCP-Cy5.5PerCP/PE-Cy5/PerCP-Cy5.5PerCP/PerCP-Cy5.5/PE-Cy5/PE-Cy5.5PE-Cy7PE-Cy7PE-Cy7APCorAlexa647APCorAlexa647APCorAlexa647APC-Cy5.5/Alexa680orAlexa700APC-Cy5.5/Alexa680orAlexa700APC-H7/APC-Cy7APC-H7/APC-Cy7APC-H7/APC-Cy7BV421HorizonV450/V500PacificOrange,Q-dots1表达强弱-AntigenDensity染料选择2•荧光染料的强度CD5CD8染料选择2Example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/cell2Eight-colorantibodypanelsproposedbytheHumanImmunophenotypingConsortiumNatRevImmunol,2012TheimportanceofantibodychoiceThestainingpatternsoftwocommerciallyavailableclonesofhumanCD38-specificantibodyareverydifferent,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spectraviewer3光谱重叠会导致数据丢失CD45-FITCDimCD4-PECD45FITC漏到PE，CD4PE弱信号分不开CD45-PerCPDimCD4-PECD45PerCP不漏到PE，弱CD4可以分开3UncompensatedCompensateddataspreadduetospillover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/cell3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:CD19FITCOnlyanissuewhenthetwomarkers(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-dye4BDHorizonTMV500CD45BDTMAPC-cy7CD14FITCCD8PerCP-CyTM5.5CD4BDHorizonTMV450CD3APCCD19PE-Cy™7CD56PECD80FluorochromeAntigenBDHorizonTMV500CD45BDTMAPC-cy7CD14FITCCD8PerCP-CyTM5.5CD4BDHorizonTMV450CD3APCCD19PE-Cy™7CD56PECD80FluorochromeAntigen不同细胞不同细胞由于tandem-dye降解会产生加阳性A.FalsepositivesinAPCchannelreducedinabsenceofAPC-Cy7FalsepositivesinPEchannelremainCD8APC-Cy7+cellsCD4PE-Cy7+cellsB.WithCD8APC-Cy7andCD4PE-Cy7WithoutCD8APC-Cy74补偿:Tandems0hours2hours22.5hoursPECD8CD3PE-Cy5PE-Cy7样本曝光时间4PerCPNewTandemsAreMoreStableAPC-H7toreplaceAPC-Cy7:ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy74•自发荧光多的选择用红激光染料选择BDHorizonTMV500CD45BDTMAPC-H7CD14FITCCD8PerCP-CyTM5.5CD4BDHorizonTMV450CD3APCCD19PE-Cy™7CD56PECD80FluorochromeAntigenPerfect!5Identificationofimmunecellsubsetsbyeight-colourantibodystainingNatRevImmunol,2012Identificationofimmunecellsubsetsbyeight-colourantibodystainingNatRevImmunol,2012Eight-colorforhematopoieticstemcellMPPs:multipotentprogenitorcellsCMPs:commonmyeloidprogenitorcellsCLPs:commonlymphoidprogenitorcellsMEPs:megakaryocyteerythroidprogenitorcellsGMPs:granulocytemacrophageprogenitorcellsEight-colorforhematopoieticstemcellEight-colorforhematopoieticstemcellSix-colorforhematopoieticstemcellBuffer选择•Fixationbuffer•BDCytofix/Cytoperm™andBD™Perm/Washbuffer•BDPharmingen™FoxP3bufferset(mouseorhuman)•BD™PhosflowPermBufferII•BD™PhosflowPermBufferIII•BDIntraSure™kitEffectofBDCytofix/CytopermBufferonMouseFoxp3StainingMouseFoxp3AlexaFluor®647Foxp3BufferCD4PEBDCytofix/Cytoperm背景处理TheImmunoglobulin背景处理-Blocking•FcBlock•MouseFcBlock,purifiedCD16/32cat#553141/553142•ReducesBackgroundStainingNopre-inc.FcBlockPre-inc.FcBlock多色应用工具123PE-CF594激发Laser：488nm或561nm发射波：612nm更亮更稳定溢漏更少FITCPEPE-CF594PerCPPE-CY7更亮的PE-CF594更稳定的PE-CF594•PE-CF594reagentshaveconsistentspillovervaluesbetweenlotsandspecificities,minimizingtheneedforlotspecificcompensationcontrolsBrilliantViolet™421BrilliantViolet™421•ExMax:407nm•EmMax:421nmand448nm用标准的HorizonV450filter:450/50nmBrilliantViolet™421：极亮极亮：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-Cy5.5CD279PEBrilliantViolet™42120%26%31%CD18444%79%TregPanel12CD127A647CD25BV421CD25PECD127BV421CD127vsCD25溢漏更少BV421V450V500稳定：StabilityinPFAfixativesSamplePrepTestingCriteriaStabilityResultsFixcells&storeinStainBuffer@4CinthedarkAbilitytoresolvepositive&negativepopulations≥96hoursStainIndexdoesnotchangemorethan25%ofTime072hoursFixcells&storeinFixative@4CinthedarkAbilitytoresolvepositive&negativepopulations≥96hoursStainIndexdoesnotchangemorethan25%ofTime048hours(1%PFA)24hours(4%PFA)BV421特点总结•极亮•溢漏更少•稳定应用：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APC-H7APC-H7ComparisonofSampleStability(inBDStabilizingFixativeatRT)0501001502002500124682448Hoursoflightexposure%SpilloverCD4APC-H7CD8APC-H7CD4APC-Cy7CD8APC-Cy7BDHorizonV450，BDHorizonV500BDHorizonV450，BDHorizonV500V450----PacificBlueV500----PacificOrangeAmCyanApoptosis,DNADamageandCellProliferationKittheApoptosis,DNADamageandCellProliferationKit•同一管分析凋亡、DNA损伤和细胞增殖•Apoptosis-cleavedPARP(PE)•DNADamage-H2AX(AlexaFluor®6