《流式细胞(FACS)多色组合原则和工具及新染料介绍.ppt》由会员分享,可在线阅读,更多相关《流式细胞(FACS)多色组合原则和工具及新染料介绍.ppt(63页珍藏版)》请在得力文库 - 分享文档赚钱的网站上搜索。
1、多色组合原则和工具新染料介绍李彩霞李彩霞 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-colorAdditionalFITC or Alexa 488FITC or Alexa 488FITC or Alexa 488PEPEPEPE-CF594 or PE-Texas Red or PE-Alexa 610/594PE-CF594 or PE-Texas Red or PE-Alexa 610/594PerCP/PE-Cy5/PerCP-Cy5.
2、5PerCP/PE-Cy5/PerCP-Cy5.5PerCP/PerCP-Cy5.5/PE-Cy5/PE-Cy5.5PE-Cy7PE-Cy7PE-Cy7APC or Alexa 647APC or Alexa 647APC or Alexa 647APC-Cy5.5/Alexa 680 or Alexa 700APC-Cy5.5/Alexa 680 or Alexa 700APC-H7/APC-Cy7APC-H7/APC-Cy7APC-H7/APC-Cy7BV 421Horizon V450/V500Pacific Orange,Q-dots1 1表达强弱-Antigen Density染料选
3、择染料选择2 2荧光染料的强度CD5CD8染料选择染料选择2 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”Pacific BlueCD7“less bright”PECD8=90K molecules/cellCD7=20K molecules/cell2 2Eight color antibody panels proposed by the Human Immunophenotyping Consortium Nat Rev Immunol,2012The importance of antibody choice
4、The staining patterns of two commercially available clones of human CD38specific antibody are very different,despite the fact that both antibodies were conjugated to allophycocyanin(APC)by the same vendor,and were used to stain peripheral blood mononuclear cells(PBMCs)from the same healthy subject u
5、nder identical conditions.V450,violet 450.Data courtesy of Angelique Biancotto,National Heart,Lung and Blood Institute,USA.Nat Rev Immunol,2012荧光染料光谱重叠最小化 spectra viewer3 3光谱重叠会导致数据丢失CD45-FITCDim CD4-PECD45 FITC 漏到 PE,CD4 PE 弱信号分不开CD45-PerCPDim CD4-PECD45 PerCP 不漏到 PE,弱 CD4 可以分开3 3UncompensatedCompe
6、nsateddata spread due to spillover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“less bright”PEBoth antigens expressed on same cell,low spillover of CD3 into CD7 and vice versa.CD3=124K molecules/cellCD7=20K molecules/cell3 3双激发荧光染料荧光染料可被不止1 laser激发,导致光谱重叠干扰,影响结果.AmCyan 可被 Violet(405nm)
7、和Blue(488nm)(FITC detector).PE-Cy5 可漏到 APC detector.Without CD45 AmCyan:With CD45 AmCyan:CD19 FITCOnly an issue when the two markers(CD45 and CD19)are co-expressed on the same cell population.3 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-dye4 4BD HorizonTMV500CD45BDTM APC-cy7CD14FITCCD8PerCP
8、-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigenBD HorizonTM V500CD45BDTM APC-cy7CD14FITCCD8PerCP-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigen不同细胞不同细胞由于 tandem-dye 降解会产生加阳性A.False positives inAPC channel reducedin absence of APC-Cy7False positives
9、in PE channelremainCD8 APC-Cy7+cellsCD4 PE-Cy7+cellsB.With CD8 APC-Cy7 and CD4 PE-Cy7Without CD8 APC-Cy74 4补偿:Tandems0 hours2 hours22.5 hoursPECD8CD3PE-Cy5PE-Cy7样本曝光时间4 4PerCPNew Tandems Are More StableAPC-H7 to replace APC-Cy7:Comparison of Sample Stability(in BD Stabilizing Fixative at RT)05010015
10、02002500124682448Hours of light exposure%SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy74 4自发荧光多的选择用红激光 染料选择BD HorizonTMV500CD45BDTM APC-H7CD14FITCCD8PerCP-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigenPerfect!5 5Identification of immune cell subsets by eight colour ant
11、ibody staining Nat Rev Immunol,2012Identification of immune cell subsets by eight colour antibody staining Nat Rev Immunol,2012Eight-color for hematopoietic stem cell MPPs:multipotent progenitor cellsCMPs:common myeloid progenitor cellsCLPs:common lymphoid progenitor cellsMEPs:megakaryocyte erythroi
12、d progenitor cells GMPs:granulocyte macrophage progenitor cellsEight-color for hematopoietic stem cellEight-color for hematopoietic stem cellSix-color for hematopoietic stem cellBuffer 选择Fixation bufferBD Cytofix/Cytoperm and BD Perm/Wash bufferBD Pharmingen FoxP3 buffer set(mouse or human)BD Phosfl
13、ow Perm Buffer IIBD Phosflow Perm Buffer IIIBD IntraSure kit Effect of BD Cytofix/Cytoperm Buffer on Mouse Foxp3 StainingMouse Foxp3 Alexa Fluor 647Foxp3 BufferCD4 PEBD Cytofix/Cytoperm背景处理The Immunoglobulin背景处理-BlockingFcBlock Mouse FcBlock,purified CD16/32 cat#553141/553142Reduces Background Stain
14、ingNo pre-inc.FcBlock Pre-inc.FcBlock多色应用工具多色应用工具123PE-CF594激发Laser:488nm 或561nm发射波:612 nml 更亮l 更稳定l 溢漏更少FITCPEPE-CF594PerCPPE-CY7更亮的PE-CF594更稳定的PE-CF594PE-CF594 reagents have consistent spillover values between lots and specificities,minimizing the need for lot specific compensation controlsBrillia
15、nt Violet 421Brilliant Violet 421 Brilliant Violet 421Ex Max:407 nmEm Max:421nm and 448 nm用标准的Horizon V450 filter:450/50 nmBrilliant Violet 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-Cy5.5CD279PEBrilliant Violet42120%26%31%CD18444%79%Treg Panel 1 2CD127 A647CD25 BV421CD25 PECD127 BV421CD127 vs CD25溢漏更少
16、BV421V450V500稳定:Stability in PFA fixativesSample PrepTesting CriteriaStability ResultsFix cells&store in Stain Buffer 4C in the darkAbility to resolve positive&negative populations 96 hoursStain Index does not change more than 25%of Time 072 hoursFix cells&store in Fixative 4C in the darkAbility to
17、resolve positive&negative populations 96 hoursStain Index does not change more than 25%of Time 048 hours(1%PFA)24 hours(4%PFA)BV421 特点总结 极亮溢漏更少稳定应用:MulticolorBV421是多色组合的理想选择尤其是弱表达/低表达Panels(FACSVerse)很亮的很亮的10色组合色组合APC-H7APC-H7Comparison of Sample Stability(in BD Stabilizing Fixative at RT)0501001502
18、002500124682448Hours of light exposure%SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy7BD Horizon V450,BD Horizon V500BD Horizon V450,BD Horizon V500V450-Pacific BlueV500-Pacific Orange AmCyanApoptosis,DNA Damage Apoptosis,DNA Damage and Cell Proliferation Kitand Cell Proliferation Kitthe Apoptos
19、is,DNA Damage and Cell Proliferation Kit同一管分析凋亡、DNA损伤和细胞增殖Apoptosis-cleaved PARP(PE)DNA Damage-H2AX(Alexa Fluor 647)Cell Proliferation BrdU(PerCP-Cy5.5)省时、节约珍贵样本Sample DataWith camptothecin treatment(bottom)cell proliferation goes down(BrdU+cells),DNA damage goes up(H2AX+cells),and apoptosis goes up(cleaved PARP+)ViabilityViability:Fixable Viability Fixable Viability Stain 450Stain 450(Cat.no.562247)Cat.no.562247)BD Horizon Fixable Viability Stain 450(FVS450)区分死活细胞Ex Max:405 nm Em Max:450 nm李彩霞李彩霞
限制150内