流式細(xì)胞(FACS)多色組合原則和工具及新染料介紹

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(yáng)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 lessbright PECD8 90Kmolecules cellCD7 20Kmolecules cell 2 Eight colorantibodypanelsproposedbytheHumanImmunophenotypingConsortium NatRevImmunol 2012 Theimportanceofantibodychoice ThestainingpatternsoftwocommerciallyavailableclonesofhumanCD38 specificantibodyareverydifferent despitethefactthatbothantibodieswereconjugatedtoallophycocyanin APC bythesamevendor andwereusedtostainperipheralbloodmononuclearcells PBMCs fromthesamehealthysubjectunderidenticalconditions V450 violet450 DatacourtesyofAngeliqueBiancotto NationalHeart LungandBloodInstitute USA NatRevImmunol 2012 熒光染料光譜重疊最小化 spectraviewer 3 光譜重疊會(huì)導(dǎo)致數(shù)據(jù)丟失 CD45 FITCDimCD4 PE CD45FITC漏到PE CD4PE弱信號(hào)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 lessbright 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(huì)產(chǎn)生加陽(yáng)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(shí)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 Identificationofimmunecellsubsetsbyeight colourantibodystaining NatRevImmunol 2012 Identificationofimmunecellsubsetsbyeight colourantibodystaining NatRevImmunol 2012 Eight colorforhematopoieticstemcell MPPs multipotentprogenitorcellsCMPs commonmyeloidprogenitorcellsCLPs commonlymphoidprogenitorcellsMEPs megakaryocyteerythroidprogenitorcellsGMPs granulocytemacrophageprogenitorcells Eight colorforhematopoieticstemcell Eight colorforhematopoieticstemcell Six colorforhematopoieticstemcell Buffer選擇 FixationbufferBDCytofix Cytoperm andBD Perm WashbufferBDPharmingen FoxP3bufferset mouseorhuman BD PhosflowPermBufferIIBD PhosflowPermBufferIIIBDIntraSure kit EffectofBDCytofix CytopermBufferonMouseFoxp3Staining MouseFoxp3AlexaFluor 647 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 1 2 3 新染料 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 更亮的PE CF594 更穩(wěn)定的PE CF594 PE CF594reagentshaveconsistentspillovervaluesbetweenlotsandspecificities minimizingtheneedforlotspecificcompensationcontrols BrilliantViolet 421 BrilliantViolet 421 ExMax 407nmEmMax 421nmand448nm用標(biāo)準(zhǔn)的HorizonV450filter 450 50nm BrilliantViolet 421 極亮 極亮 更好的細(xì)胞分群 亮的染料使細(xì)胞分群更明顯陽(yáng)性率增加 PerCP Cy5 5 CD279 PE BrilliantViolet 421 20 26 31 CD184 44 79 TregPanel12 CD127A647 CD25BV421 CD25PE CD127BV421 CD127vsCD25 溢漏更少 BV421 V450 V500 穩(wěn)定 StabilityinPFAfixatives BV421特點(diǎn)總結(jié) 極亮溢漏更少穩(wěn)定 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 APC H7 APC H7 ComparisonofSampleStability inBDStabilizingFixativeatRT 0 50 100 150 200 250 0 1 2 4 6 8 24 48 Hoursoflightexposure Spillover BDHorizonV450 BDHorizonV500 BDHorizonV450 BDHorizonV500 V450 PacificBlueV500 PacificOrangeAmCyan 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 AlexaFluor 647 CellProliferation BrdU PerCP Cy5 5 省時(shí) 節(jié)約珍貴樣本 SampleData Withcamptothecintreatment bottom cellproliferationgoesdown BrdU ce