流式细胞FACS多色组合原则和工具及新染料介绍ppt课件.ppt

多色组合原则和工具新染料介绍 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 Eight colorantibodypanelsproposedbytheHumanImmunophenotypingConsortium NatRevImmunol 2012 7 Theimportanceofantibodychoice ThestainingpatternsoftwocommerciallyavailableclonesofhumanCD38 specificantibodyareverydifferent despitethefactthatbothantibodieswereconjugatedtoallophycocyanin APC bythesamevendor andwereusedtostainperipheralbloodmononuclearcells PBMCs fromthesamehealthysubjectunderidenticalconditions V450 violet450 DatacourtesyofAngeliqueBiancotto NationalHeart LungandBloodInstitute USA NatRevImmunol 2012 8 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 Identificationofimmunecellsubsetsbyeight colourantibodystaining NatRevImmunol 2012 18 Identificationofimmunecellsubsetsbyeight colourantibodystaining NatRevImmunol 2012 19 Eight colorforhematopoieticstemcell MPPs multipotentprogenitorcellsCMPs commonmyeloidprogenitorcellsCLPs commonlymphoidprogenitorcellsMEPs megakaryocyteerythroidprogenitorcellsGMPs granulocytemacrophageprogenitorcells 20 Eight colorforhematopoieticstemcell 21 Eight colorforhematopoieticstemcell 22 Six colorforhematopoieticstemcell 23 Buffer选择 FixationbufferBDCytofix Cytoperm andBD Perm WashbufferBDPharmingen FoxP3bufferset mouseorhuman BD PhosflowPermBufferIIBD PhosflowPermBufferIIIBDIntraSure kit EffectofBDCytofix CytopermBufferonMouseFoxp3Staining MouseFoxp3AlexaFluor 647 Foxp3Buffer CD4PE BDCytofix Cytoperm 背景处理 TheImmunoglobulin 背景处理 Blocking FcBlockMouseFcBlock purifiedCD16 32cat 553141 553142ReducesBackgroundStaining Nopre inc FcBlockPre inc FcBlock 多色应用工具 28 1 29 2 30 3 31 32 33 34 35 36 37 38 39 40 41 42 新染料 43 PE CF594 激发Laser 488nm或561nm发射波 612nm 更亮更稳定溢漏更少 FITCPEPE CF594PerCPPE CY7 44 更亮的PE CF594 45 更稳定的PE CF594 PE CF594reagentshaveconsistentspillovervaluesbetweenlotsandspecificities minimizingtheneedforlotspecificcompensationcontrols 46 BrilliantViolet 421 BrilliantViolet 421 ExMax 407nmEmMax 421nmand448nm用标准的HorizonV450filter 450 50nm 48 BrilliantViolet 421 极亮 49 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 BrilliantViolet 421 20 26 31 CD184 44 79 50 TregPanel12 CD127A647 CD25BV421 CD25PE CD127BV421 CD127vsCD25 51 溢漏更少 BV421 V450 V500 52 稳定 StabilityinPFAfixatives 53 BV421特点总结 极亮溢漏更少稳定 54 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 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损伤和细胞增殖Apoptosis cleavedPARP PE DNADamage H2AX AlexaFluor 647 CellProliferation BrdU PerCP Cy5 5 省时 节约珍贵样本 59 SampleData Withcamptothecintreatment bottom cellproliferationgoesdown BrdU cells DNAdamagegoesup