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1、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。流式细胞FACS多色组合原则和工具及新染料介绍染料选择-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/P
2、E-Cy5/PerCP-Cy5.5PerCP/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流式细胞FA
3、CS多色组合原则和工具及新染料介绍表达强弱- Antigen Density染料选择染料选择2 2流式细胞FACS多色组合原则和工具及新染料介绍荧光染料的强度CD5CD8染料选择染料选择2 2流式细胞FACS多色组合原则和工具及新染料介绍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” Pacific BlueCD7 “less bright” PECD8 = 90K molecules/cellCD7 = 20K molecules/cell2 2流式细胞FACS多色组合原则和工具及新染料介绍Eight color an
4、tibody panels proposed by the Human Immunophenotyping Consortium Nat Rev Immunol, 20127流式细胞FACS多色组合原则和工具及新染料介绍The importance of antibody choice The staining patterns of two commercially available clones of human CD38specific antibody are very different, despite the fact that both antibodies were con
5、jugated to allophycocyanin (APC) by the same vendor, and were used to stain peripheral blood mononuclear cells (PBMCs) from the same healthy subject under identical conditions. V450, violet 450. Data courtesy of Angelique Biancotto, National Heart, Lung and Blood Institute, USA. Nat Rev Immunol, 201
6、28流式细胞FACS多色组合原则和工具及新染料介绍荧光染料光谱重叠最小化 spectra viewer3 3流式细胞FACS多色组合原则和工具及新染料介绍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 3UncompensatedCompensateddata spread due to spillover流式细胞FACS多色组合原则和工具及新染料介绍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Exam
7、ple: 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流式细胞FACS多色组合原则和工具及新染料介绍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) 和Blue（488nm) (FITC detector).PE-Cy
8、5 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流式细胞FACS多色组合原则和工具及新染料介绍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-CyTM5.
9、5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigenBD HorizonTM V500CD45BDTM APC-cy7CD14FITCCD8PerCP-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD80FluorochromeAntigen不同细胞不同细胞流式细胞FACS多色组合原则和工具及新染料介绍由于 tandem-dye 降解会产生加阳性A.False positives inAPC channel reducedin absence of APC-Cy
10、7False positivesin PE channelremainCD8 APC-Cy7+ cellsCD4 PE-Cy7+ cellsB.With CD8 APC-Cy7 and CD4 PE-Cy7Without CD8 APC-Cy74 4流式细胞FACS多色组合原则和工具及新染料介绍补偿: Tandems0 hours2 hours22.5 hoursPECD8CD3PE-Cy5PE-Cy7样本曝光时间4 4PerCP流式细胞FACS多色组合原则和工具及新染料介绍New Tandems Are More StableAPC-H7 to replace APC-Cy7:Compari
11、son of Sample Stability(in BD Stabilizing Fixative at RT)0501001502002500124682448Hours of light exposure% SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy74 4流式细胞FACS多色组合原则和工具及新染料介绍自发荧光多的选择用红激光 染料选择BD HorizonTMV500CD45BDTM APC-H7CD14FITCCD8PerCP-CyTM5.5CD4BD HorizonTM V450CD3APCCD19PE-Cy7CD56PECD
12、80FluorochromeAntigenPerfect!5 5流式细胞FACS多色组合原则和工具及新染料介绍Identification of immune cell subsets by eight colour antibody staining Nat Rev Immunol,201218流式细胞FACS多色组合原则和工具及新染料介绍Identification of immune cell subsets by eight colour antibody staining Nat Rev Immunol,201219流式细胞FACS多色组合原则和工具及新染料介绍Eight-color
13、 for hematopoietic stem cell MPPs: multipotent progenitor cellsCMPs: common myeloid progenitor cellsCLPs: common lymphoid progenitor cellsMEPs: megakaryocyte erythroid progenitor cells GMPs: granulocyte macrophage progenitor cells20流式细胞FACS多色组合原则和工具及新染料介绍Eight-color for hematopoietic stem cell21流式细胞
14、FACS多色组合原则和工具及新染料介绍Eight-color for hematopoietic stem cell22流式细胞FACS多色组合原则和工具及新染料介绍Six-color for hematopoietic stem cell23流式细胞FACS多色组合原则和工具及新染料介绍Buffer 选择Fixation bufferBD Cytofix/Cytoperm and BD Perm/Wash bufferBD Pharmingen FoxP3 buffer set (mouse or human)BD Phosflow Perm Buffer IIBD Phosflow Per
15、m Buffer IIIBD IntraSure kit流式细胞FACS多色组合原则和工具及新染料介绍 Effect of BD Cytofix/Cytoperm Buffer on Mouse Foxp3 StainingMouse Foxp3 Alexa Fluor 647Foxp3 BufferCD4 PEBD Cytofix/Cytoperm流式细胞FACS多色组合原则和工具及新染料介绍背景处理The Immunoglobulin流式细胞FACS多色组合原则和工具及新染料介绍背景处理-BlockingFcBlock Mouse FcBlock, purified CD16/32 cat
16、 # 553141/553142Reduces Background StainingNo pre-inc. FcBlock Pre-inc. FcBlock流式细胞FACS多色组合原则和工具及新染料介绍多色应用工具多色应用工具28流式细胞FACS多色组合原则和工具及新染料介绍129流式细胞FACS多色组合原则和工具及新染料介绍230流式细胞FACS多色组合原则和工具及新染料介绍331流式细胞FACS多色组合原则和工具及新染料介绍32流式细胞FACS多色组合原则和工具及新染料介绍33流式细胞FACS多色组合原则和工具及新染料介绍34流式细胞FACS多色组合原则和工具及新染料介绍35流式细胞FA
17、CS多色组合原则和工具及新染料介绍36流式细胞FACS多色组合原则和工具及新染料介绍37流式细胞FACS多色组合原则和工具及新染料介绍38流式细胞FACS多色组合原则和工具及新染料介绍39流式细胞FACS多色组合原则和工具及新染料介绍40流式细胞FACS多色组合原则和工具及新染料介绍41流式细胞FACS多色组合原则和工具及新染料介绍42流式细胞FACS多色组合原则和工具及新染料介绍43流式细胞FACS多色组合原则和工具及新染料介绍PE-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-CY744流式
18、细胞FACS多色组合原则和工具及新染料介绍更亮的PE-CF59445流式细胞FACS多色组合原则和工具及新染料介绍更稳定的PE-CF594PE-CF594 reagents have consistent spillover values between lots and specificities, minimizing the need for lot specific compensation controls46流式细胞FACS多色组合原则和工具及新染料介绍Brilliant Violet 421 流式细胞FACS多色组合原则和工具及新染料介绍Brilliant Violet 421E
19、x Max: 407 nmEm Max: 421nm and 448 nm用标准的Horizon V450 filter: 450/50 nm48流式细胞FACS多色组合原则和工具及新染料介绍Brilliant Violet 421 ：极亮49流式细胞FACS多色组合原则和工具及新染料介绍极亮：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%50流式细胞FACS多色组合原则和工具及新染料介绍 Treg Panel 1 2CD127 A647CD25 BV421CD25 PECD1
20、27 BV421CD127 vs CD2551流式细胞FACS多色组合原则和工具及新染料介绍溢漏更少BV421V450V50052流式细胞FACS多色组合原则和工具及新染料介绍稳定 ：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 mor
21、e than 25% of Time 072 hoursFix cells & store in Fixative 4C in the darkAbility to resolve positive & negative populations 96 hoursStain Index does not change more than 25% of Time 048 hours (1% PFA)24 hours (4% PFA)53流式细胞FACS多色组合原则和工具及新染料介绍BV421 特点总结 极亮溢漏更少稳定54流式细胞FACS多色组合原则和工具及新染料介绍应用：MulticolorBV
22、421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55流式细胞FACS多色组合原则和工具及新染料介绍APC-H7APC-H7Comparison of Sample Stability(in BD Stabilizing Fixative at RT)0501001502002500124682448Hours of light exposure% SpilloverCD4 APC-H7CD8 APC-H7CD4 APC-Cy7CD8 APC-Cy756流式细胞FACS多色组合原则和工具及新染料介绍BD Horizon V450， B
23、D Horizon V500BD Horizon V450， BD Horizon V500V450- Pacific BlueV500- Pacific Orange AmCyan57流式细胞FACS多色组合原则和工具及新染料介绍Apoptosis, DNA Damage and Cell Proliferation Kit流式细胞FACS多色组合原则和工具及新染料介绍the Apoptosis, 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- H2A
24、X (Alexa Fluor 647)Cell Proliferation BrdU (PerCP-Cy5.5)省时、节约珍贵样本59流式细胞FACS多色组合原则和工具及新染料介绍Sample DataWith camptothecin treatment (bottom) cell proliferation goes down (BrdU+ cells), DNA damage goes up (H2AX+ cells), and apoptosis goes up (cleaved PARP+)60流式细胞FACS多色组合原则和工具及新染料介绍Viability：Fixable Viability Stain 450（Cat.no. 562247) 流式细胞FACS多色组合原则和工具及新染料介绍BD Horizon Fixable Viability Stain 450 (FVS450)区分死活细胞Ex Max: 405 nm Em Max: 450 nm62流式细胞FACS多色组合原则和工具及新染料介绍李彩霞李彩霞 63流式细胞FACS多色组合原则和工具及新染料介绍
