流式细胞(FACS)多色组合原则和工具及新染料介绍.ppt
多色组合原则和工具新染料介绍,李彩霞 13910675952 Caixia_,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- Antigen Density,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” Pacific Blue CD7 “less bright” PE CD8 = 90K molecules/cell CD7 = 20K molecules/cell,2,Eightcolor antibody panels proposed by the Human Immunophenotyping Consortium,Nat Rev Immunol, 2012,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 conjugated 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, 2012,荧光染料光谱重叠最小化,spectra viewer,3,光谱重叠会导致数据丢失,CD45-FITC Dim CD4-PE,CD45 FITC 漏到 PE， CD4 PE 弱信号分不开,CD45- PerCP Dim CD4-PE,CD45 PerCP 不漏到 PE ，弱 CD4 可以分开,3,Uncompensated,Compensated,data 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-Cy7 CD7 “less bright” PE Both antigens expressed on same cell, low spillover of CD3 into CD7 and vice versa. CD3 = 124K molecules/cell CD7 = 20K molecules/cell,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) 和Blue（488nm) (FITC detector). PE-Cy5 可漏到 APC detector.,Without CD45 AmCyan:,With CD45 AmCyan:,CD19 FITC,Only an issue when the two markers (CD45 and CD19) are co-expressed on the same cell population.,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.,False positives in APC channel reduced in absence of APC-Cy7,False positives in PE channel remain,CD8 APC-Cy7+ cells,CD4 PE-Cy7+ cells,B.,With CD8 APC-Cy7 and CD4 PE-Cy7,Without CD8 APC-Cy7,4,补偿: Tandems,0 hours,2 hours,22.5 hours,PE,CD8,CD3,PE-Cy5,PE-Cy7,样本曝光时间,4,PerCP,New Tandems Are More Stable,APC-H7 to replace APC-Cy7:,Comparison of Sample Stability,(in BD Stabilizing Fixative at RT),0,50,100,150,200,250,0,1,2,4,6,8,24,48,Hours of light exposure,% Spillover,4,自发荧光多的选择用红激光,染料选择,Perfect!,5,Identification of immune cell subsets by eightcolour antibody staining,Nat Rev Immunol,2012,Identification of immune cell subsets by eightcolour antibody staining,Nat Rev Immunol,2012,Eight-color for hematopoietic stem cell,MPPs: multipotent progenitor cells CMPs: common myeloid progenitor cells CLPs: common lymphoid progenitor cells MEPs: megakaryocyte erythroid progenitor cells GMPs: granulocyte macrophage progenitor cells,Eight-color for hematopoietic stem cell,Eight-color for hematopoietic stem cell,Six-color for hematopoietic stem cell,Buffer 选择,Fixation buffer BD Cytofix/Cytoperm and BD Perm/Wash buffer BD Pharmingen FoxP3 buffer set (mouse or human) BD Phosflow Perm Buffer II BD Phosflow Perm Buffer III BD IntraSure kit,Effect of BD Cytofix/Cytoperm Buffer on Mouse Foxp3 Staining,Mouse Foxp3 Alexa Fluor 647,Foxp3 Buffer,CD4 PE,BD Cytofix/Cytoperm,背景处理,The Immunoglobulin,背景处理-Blocking,FcBlock Mouse FcBlock, purified CD16/32 cat # 553141/553142 Reduces Background Staining,No pre-inc. FcBlock Pre-inc. FcBlock,多色应用工具,1,2,3,新染料,PE-CF594,激发Laser：488nm 或561nm 发射波： 612 nm,更亮 更稳定 溢漏更少,FITC PE PE-CF594 PerCP PE-CY7,更亮的PE-CF594,更稳定的PE-CF594,PE-CF594 reagents have consistent spillover values between lots and specificities, minimizing the need for lot specific compensation controls,Brilliant Violet 421,Brilliant Violet 421,Ex Max: 407 nm Em Max: 421nm and 448 nm 用标准的Horizon V450 filter: 450/50 nm,Brilliant 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-Cy5.5,CD279,PE,Brilliant Violet421,20%,26%,31%,CD184,44%,79%,Treg Panel 1 2,CD127 A647,CD25 BV421,CD25 PE,CD127 BV421,CD127 vs CD25,溢漏更少,BV421,V450,V500,稳定 ：Stability in PFA fixatives,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-H7,APC-H7,Comparison of Sample Stability,(in BD Stabilizing Fixative at RT),0,50,100,150,200,250,0,1,2,4,6,8,24,48,Hours of light exposure,% Spillover,BD Horizon V450， BD Horizon V500,BD Horizon V450， BD Horizon V500,V450- Pacific Blue V500- Pacific Orange AmCyan,Apoptosis, DNA Damage and Cell Proliferation Kit,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- H2AX (Alexa Fluor 647) Cell Proliferation BrdU (PerCP-Cy5.5) 省时、节约珍贵样本,Sample Data,With camptothecin treatment (bottom) cell proliferation goes down (BrdU+ cells), DNA damage goes up (H2AX+ cells), and apoptosis goes up (cleaved PARP+),Viability：Fixable Viability Stain 450（Cat.no. 562247),BD Horizon Fixable Viability Stain 450 (FVS450),区分死活细胞,Ex Max: 405 nm Em Max: 450 nm,Thanks,李彩霞 13910675952 Caixia_,
