流式细胞术基本原理及应用.ppt

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,流式细胞术原理及应用,For FCM Training,流式细胞术,流式细胞术（,Flow Cytometry,简称,FCM,）是一种可以快速、准确、客观，并且同时检测单个微粒（通常是细胞）的多项特性的技术，同时可以对特定群体加以分选,研究对象为生物颗粒，如各种细胞、染色体、微生物、及人工合成微球等,研究的微粒特性包括多种物理及生物学特征，并加以定量,流式细胞仪系统,简介,通过流式细胞仪我们可以得到以下信息,-,相对细胞大小,-,相对细胞颗粒密度和内部复杂度,-,染色过细胞的相对荧光强度,Injector,Tip,Fluorescence,signals,Focused laser,beam,Sheath,fluid,流式细胞仪的光信号,散射光信号,荧光信号,1.,散射光信号,前向角散射光（,FSC,Forward Scatter,）,入射激光的同向散射光信号,细胞相 对大小及其表面积。,侧向角散射（,SSC,Side Scatter,）,入射激光,90,角的,散射光信号,细胞粒度及细胞内相对复杂性。,前向角散射光,FSC,Forward Angle Light Scatter,FALS Sensor,Laser,侧向角散射光,SSC,FALS Sensor,90LS Sensor,Laser,散射光,散射光能被用来区分不同细胞群体的基本形态上的差异,-,通常使用,“,散点图,”,来看散射光信号,-,散点图上的一个点就代表一个细胞颗粒的数据,散点图,Dot Plot,lysed whole blood,Review Question,Dead cells are known to be smaller and to exhibit more internal complexity than live cells.Which of the populations on this plot would you expect to be dead?,A,B,2.,荧光信号,荧光素吸收激光能量,荧光素将吸收能量释放，转换为,振动能和热能,释放较入射光波长更长的光量子,荧光素与特异抗体结合,荧光抗体与细胞抗原结合越多，产生的荧光信号越强,荧光检测器,Fluorescence,FALS Sensor,Fluorescence detector,(PMT3,PMT4 etc.),Two-Color Cell Analysis,Which of the three populations has the most Ab A binding sites?,Ab A,Ab B,双色荧光散点图,现代流式细胞仪包括,液流系统,聚焦细胞以供检测,光学系统,激发和收集光信号,电子系统,将光信号转化为电信号，并使其数字化以供计算机分析,液流系统,液流系统将样本悬液聚焦在光源的中心处,Sample Flow in Optical Cuvette,Sample,LaminarFlow,Low Sample Flow Rate,12,m,L/min,Sheath,Sheath,Sample,LaminarFlow,Sheath,Sheath,Optical Cuvette,High Sample Flow Rate,60,m,L/min,Review Question,Which of the following would cause disturbance in the laminar flow of the optical cuvette?,bubbles,cellular concentration,sample flow rate,Optics,Excitation optics consist of:,Lasers,Lenses and mirrors that route the laser light to the fluidic stream,Collection optics consist of:,Filters that direct the signals to the appropriate optical detectors,Optical Filters,460 500 540,460 500 540,460 500 540,SP 500,LP 500,BP500/50,LongpassShortpassBandpass,FACSCalibur,光路图,Optics,Laser,Fluorochromes,Detector Parameter,Filter,FITC,GFP,FL1,530/30,PE,PI,FL2,585/42,PerCP,PerCP-CY5.5,FL3,670LP,APC,FL4,661/16,488 nm,635 nm,Electronics,Converts analog signals to proportional digital signals,Computes Height,for each pulse,Calculates width and area,Interfaces with the computer for data transfer,Creation of a Voltage Pulse-Analog Signal,Laser,Laser,Laser,Time,Voltage,Time,Voltage,Time,Voltage,Quantification of a Voltage Pulse,Time,Volts,Pulse Area,Pulse Height,Pulse Width,0,Height is a measurement for all parameters.,Width=Area/Height,Effect of the Instrument Controls on the Data,Instrument Controls,Detector,FSC detector 250 gain,FSC detector 350 gain,Review,Data Processing,FL2,FL1,SSC,FSC,FL4,FL3,Review Questions,Which of the following fluorochromes cannot be used with the FACSCalibur?,DAPI(ex.345 nm,emits 455 nm),Propidium Iodide(ex.536 nm,emits 617 nm),Alexa Fluor 647(ex.650 nm,emits 668 nm),What are the three measurements of a particle that can be determined by FACSCalibur?,Briefly describe the functions of the fluidics,optics,and electronics systems.,Review Questions,What would happen to the population below if you increased the Red parameter value in the Instrument controls?,Review Questions,Which instrument components ensure that the fluorescence signal of a specific fluorochrome is only measured by a designated detector?For example,APC is only measured by the FL4 detector.,样本处理,细胞悬液的制备,细胞悬液：,分离,PBMC,、,PRP,等：操作复杂，分离、离心步骤导致细胞特定群体丢失，并可能引入某些误差,直接使用外周血、骨髓：最接近生理状况，操作简便，样本用血量小,灌洗液、体腔积液,培养细胞、细胞系,实体组织：,病理组织：新鲜样本,/,石蜡包埋样本,针吸组织：新鲜样本,鞘液、洗液等：清洁无颗粒杂质,步骤一：,选择合适的荧光染料,必须能够被流式细胞仪上所配备的激光器所激发,激发的光谱必须在仪器上滤光片能够接受的合适范围内,荧光素光谱的重叠应当尽量减少,荧光产生过程,Propidium Iodide,400 nm,500 nm,600 nm,700 nm,PI,DNA,Excitation,Emisson,300 nm 400 nm 500 nm 600 nm 700 nm,Fluorescein(FITC),400 nm,500 nm,600 nm,700 nm,Wavelength,Protein,Excitation,Emisson,300 nm 400 nm 500 nm 600 nm 700 nm,Excitation,Emisson,300 nm 400 nm 500 nm 600 nm 700 nm,Phycoerytherin(PE),Protein,Allophycocyanin(APC),Protein,632.5 nm(HeNe,),Excitation,Emisson,300 nm 400 nm 500 nm 600 nm 700 nm,FITC,和,PE,荧光光谱,补偿模型图,补偿调节前后对比,FL1,FL2,步骤二：染色,体积,温度,孵育时间,对照,直接染色,Fluorescent probe attached to antibody,Specific signal:weak,Nonspecific binding:low,间接染色,Fluorescent probe attached to a 2,nd,antibody,Specific signal:strong,5-6 2nd Ab/each 1st Ab;,Nonspecific binding:high,Avidin-Biotin method I,biotinylated primary Ab,biotin,avidin,biotinylated dye,示意图,步骤三：数据收集和分析,画图,寻找目标细胞,调整仪器设置到合适的状态,我们可以得到怎样的结果？它们意味着什么？,仪器设置调节,1.,用未染色细胞调整仪器,PMT,电压,2.,用单染色的细胞调节仪器补偿,关于同型对照,例：一个双色染色的实验,抗体,A FITC,，抗体,B PE,对应的同型对照是,IgG1 FITC,IgG1 PE,那么需要准备的是,阴性对照：细胞加上,IgG1 FITC,IgG1 PE,单阳性对照：,FITC:,细胞加上,Ab A FITC,IgG1 PE,PE:,细胞加上,Ab B PE,IgG1 FITC,数据分析,设门,设定阴性与阳性群体的界限,确定阳性与阴性细胞群体,统计阳性或阴性细胞群体的百分率，平均荧光值，绝对数或抗体结合数,如何设定阴性与阳性的界限,直方图,散点图,细胞结构,细胞大小,细胞粒度,细胞表面面积,核浆比例,DNA,含量与细胞周期,RNA,含量,蛋白质含量,染色体分析,细胞功能,细胞表面,/,胞浆,/,核的特异性抗原,细胞活性,细胞内细胞因子,酶活性,激素结合位点,细胞受体,细胞凋亡,在上述信号基础上的细胞分选,流式细胞术的细胞学应用,流式细胞仪的临床应用,HIV,免疫分型，,CD4,绝对计数,白血病和淋巴瘤的免疫分型,肿瘤的细胞周期和倍体分析,网织红细胞计数,细胞移植的交叉配型和免疫状态监测,干细胞计数,残量白血病细胞检查,HLA-B27,检查,血小板功能及相关疾病,流式细胞仪的科研应用,免疫功能研究,癌症病人的多药耐药性,细胞动力学功能研究,动物性别筛选,海洋与环境微生物分析,染色体分选,流式细胞仪的遗传学应用,细胞,DNA,RNA,含量的测定,染色体倍体分析,染色体分离,基因表达产物的生物活性研究,基因转染表达的生物效应,基因表达调控研究,细胞内基因定位,酵母转基因株的筛选,报告基因的定性定量检测,植物遗传学研究,Flow Cytometry,in clinics and research,What can Flow Cytometer tell us about a cell?,Cell Lineage/Subset Phenotype,Cytokine/Chemokine,Production Repertoire,Cytokine/Chemokine,Receptor RepertoireMigration/,Homing Phenotypre,Cell cycle status,细胞增殖（细胞周期）,细胞内,DNA,含量,增殖相关基因的表达,BrdU,的结合,示踪染料,DNA,探针,DNA,探针最主要的特点是，它们与,DNA,含量是成,化学正比,关系的,这样，带有的探针荧光分子的数目就和,DNA,分子的含量相当，从而检测,DNA,含量,Nucleic acid Probes,菲啶基,Propidium Iodide,Ethidium Bromide,苯甲亚胺,Hoechst 33342,抗生素,Mithramycin,Chromamycin A3,Acridine Orange-AO,Pyronyn Y,G,2,M,G,0,G,1,s,0,200,400,600,800,1000,G,0,G,1,s,G,2,M,DNA Analysis,DNA content,Count,2N,4N,Normal Cell Cycle,A typical DNA Histogram,G,0,-G,1,S,G,2,-M,Fluorescence Intensity,#of Events,红色为正常二倍体细胞；黄色为异倍体细胞,增殖相关基因,PCNA,(Proliferating cell nuclear antigen),一种蛋白质，在,DNA,复制和核苷酸的切除修复中起到作用,Ki-67,-proliferation related antigen,Ki-S1,-proliferation related antigen,Phospho-histone,Cyclin D1,E,A,B1,BrdUrd Incorporation,Bromodeoxyuridine(BrdU),是一个胸腺核苷的类似物,能够在细胞增殖和细胞周期状态分析中起作用,BrdU,能与正在复制周期内的细胞相结合,使用,BrdU,抗体能够检测到,BrdU,BrdUrd Incorporation,细胞分裂的分析,哺乳动物的免疫系统需要有大量的增殖，能够确保抗原特异性的,T,细胞和,B,细胞具有足够的增殖速度，能够对付治病生物造成的感染,CFSE,是一种示踪染料，能够均等地在两个子代细胞中分布，其结果可以区分出,8-10,代的子细胞。,Tracing Dye(CFSE),Apoptotic,Cell Death,-,a genetically encoded cell death program,which is morphologically,biochemically and molecularly distinct from necrosis,Flow cytometry of apoptotic cell death,细胞散射光,在细胞调亡中，细胞收缩，从而,FSC,下降，,SSC上升或是没有明显变化,Flow cytometry of apoptotic cell death,荧光吸收,细胞的胞膜对于,DNA,染料的通透性和细胞的活性、死亡和凋亡相关，像,PI,、,EB,、,Hoechst-33342,等，可以用来区分活细胞、死细胞和凋亡细胞,Flow cytometry of apoptotic cell death,FCM of Caspases,通过抗体和活化的,caspase-3,片断相结合来检测,使用特异性的,caspase-3,荧光底物,新的抗原决定基,CK18,Flow cytometry of apoptotic cell death,线粒体功能的变化,TMP,会在调亡中产生，可以通过一些标记用流式细胞仪检测。使用有膜穿透性的亲脂性阳离子荧光染料，如,Rh123,DiOC6,JC-1,CMXRos,等，可作为流式检测的探针。,当细胞发生调亡时，一个线粒体膜表面蛋白,7A6,抗原会出现,Bcl-2/bax family of proteins.,Flow cytometry of apoptotic cell death,钙离子流和,pH,值变化,胞浆内,Ca2+,水平的上升和由于细胞内环境酸化造成的选择性的,pH,值的调节降低是伴随着细胞凋亡产生的后果之一。,使用,Ca2+,选择性荧光探针，如,Quin-2,Fluo-3,Indo-1,通过流式检测是测定细胞内,Ca2+,浓度的最佳方案,酸化的检测同样可以用对,pH,敏感的荧光探针，如,DCH,BCECF,BCECF-AM,SNAFLs,SNARFs,等进行检测,Flow cytometry of apoptotic cell death,Phospholipid redistribution,Flow cytometry of apoptotic cell death,DNA,链的断裂,细胞凋亡晚期中，核酸内切酶（某些,Caspase,的底物）在核小体之间剪切核,DNA,，产生大量长度在,180-200 bp,的,DNA,片段。断裂的末端可以通过末端转脱氧核苷酰酶,(TdT)连接上,dUTP,。,Flow cytometry of apoptotic cell death,细胞,DNA,含量,在凋亡细胞中，用,PI,染色，通过流式检测,DNA,含量，可以发现一种亚群的细胞，其染色荧光强度下降。这是由于随着调亡的进行，核酸内切酶活化，随后一部分,DNA,泄漏出去，造成细胞内,DNA,含量下降造成的。,Flow Cytometry of Apoptotic Cells,PI-Fluorescence,#Events,Apoptotic cells,Normal G0/G1 cells,An Integrated Approach to Cell ImmunologyImmune Function assay,免疫细胞亚群检测,Antigen-peptide specific T cells,检测,T-cells,活化,Treg Cells,细胞内细胞因子,/,趋化因子检测,磷酸化,淋巴细胞亚群分析,根据功能，淋巴细胞主要分为,B,淋巴细胞（,CD19+），,与体液免疫有关,T,淋巴细胞（,CD3+），,与细胞免疫有关,总,T,和总,B,可以用来判断某些免疫缺陷和自身免疫性疾病,NK,细胞（,CD3-CD16+56+），,行使免疫监控功能,，,能够介导对某些肿瘤细胞和病毒感染细胞的细胞毒性作用。,淋巴细胞亚群分析,根据,CD4、CD8,表达，,T,淋巴细胞又分为,T,辅助/诱导细胞（,CD3+CD4+）,T,抑制/细胞毒性细胞（,CD3+CD8+）,Th/Ts,评价那些自身免疫失调或被怀疑是免疫失调或已知患有免疫缺陷的病人的免疫状态，此外，这一比值还可用来监测骨髓移植病人以免受到急性,GVHD,的攻击,Th/Ts,升高：自身免疫性疾病（类风湿性关节炎、,SLE）,Th/Ts,降低：病毒感染、恶性肿瘤、再生障碍性贫血,Traditional Analysis-Percent positive,Positive,cell,Negative,Cell,CD4 PE,CD4 PE,Absolute Counts-Cells per,m,L,Positive,Cell,Absolute,Count,Beads,Negative,Cell,CD4 PE,CD4 PE,抗原特异性,T,细胞,提供检测者一个强有力的工具用于研究对病毒抗原的免疫应答和疫苗的开发,MHC:Class I and Class II Gene Products,T cell,受体不直接和可溶性抗原相连,抗原必须通过,MHC,由抗原呈递细胞传递给,T,细胞,(Macrophages,Dendritic cells,B cells),MHC Class I Gene Products,呈递抗原给,CD8,+,T cells,诱导细胞毒应答,MHC Class II Gene Products,呈递抗原给,CD4,+,T cells,诱导细胞因子产生，免疫球蛋白的分泌,MHC/TCR Signaling,二聚体可溶性,MHC,类似物与四聚体的比较,Quantitation of antigen-specific T lymphocytes in peripheral blood,HAM,Control,HIV,CD8,Tax-A2/Ig,Gag-A2/Ig,Treg,细胞的检测,Treg,细胞与自身免疫耐受相关，通常的检测方法用,CD4,阳性细胞中,CD25,高表达，同时结合胞内,FoxP3,含量来判断,最新发现人的,Treg,细胞低表达,CD127,。,CD127的表达模式与,Foxp3,很接近，说明低表达,CD127,，高,/,中表达,CD25,的,CD4,阳性的,T,淋巴细胞就是,Treg,细胞（调节性,T,细胞）。该方法比胞内检测,Foxp3,的优点在于，检测后的细胞可用于后续的培养及其他的体外试验。另外的优点是用这种检测方案分选的,Treg,细胞得率更高。,Immune Function Assays-A Powerful Research Tool for Answering Basic Biological Questions in.,AIDS,或癌症机理的研究,T cell,亚群对于病毒和细菌抗原的反应,细胞介导的对机会感染的免疫反应,药物,/,疫苗的效果评价,免疫调节,移植监控,毒理研究,Traditional,Cytokine Flow Cytometry,IL-2 Phycoerythrin,CD4 FITC,细胞因子检测,细胞因子是可溶性蛋白，在淋巴细胞免疫功能调节方面发挥重要作用。细胞因子可以调节多种细胞的生长、分化和功能，调节正常与病理状态下的免疫应答，研究生理或病理免疫调节因素所致细胞因子合成的应答与改变是研究疾病病因与免疫状态的重要工具,。,T,细胞的细胞因子分型,型细胞介导免疫反应,引起迟发过敏反应、巨噬细胞活化、,2,型反应下调,刺激来源：病毒、某些细菌,型体液介导免疫反应,引起,B,细胞增殖、分泌多克隆免疫球蛋白、,1,型反应下调,刺激来源：多细胞寄生虫、过敏源,细胞因子,Biological Variation Among CMV-seropositive Donors in Response to CMV,CFC&Proliferation,使用温和的方法进行细胞破膜和固定，在中性,pH,值条件下，使细胞和,BrdU,结合，可以同时检测到：,S,期,细胞周期,表型,活化状态,核型决定基,胞浆内决定基,细胞因子,/,趋化因子,其他,.,CFC&Proliferation,Allows the correlation of:,Phenotype,Cytokine expression,Cell Cycle,Proliferation,BD Phosflow,磷酸化检测系统,更好的特异性的抗体,建立在单个细胞基础上的检测,可以同时检测多个参数,快速、灵敏、样本量更少,P,P,Y,Y,Y,Y,非磷酸化特异性抗体,检测全部的相关蛋白,unphosphorylated+phosphorylated,亚型,抗原：重组的蛋白质片断,磷酸化特异性抗体,仅仅检测磷酸化亚型,抗原：合成的,4-10mer,磷酸化肽段,Y,磷酸化特异性和非磷酸化特异性抗体的差异,BD PhosFLOW,和,Western blot,的比较,A theoretical experiment comparing Western blot and flow cytometry with three samples and a protein of interest at 1,10,or 50 copies per cell.Sample 2 and 3 look the same via Western blot,but when stained with fluorescently labeled antibodies,the differences between the samples become more relevant.(Source:P.O.Krutzik,et al,./,Clinical Immunology,110(2004)206221),BD,PhosFlow,Genomics&Proteomics,-,Single Cells Have Big Proteomics Story to,Tell.,BD PhosFlow not only tells you activation of a single cell for one particular phospho protein and pathway,but allows you to study multiple phosphorylation events and pathways simultaniously.,10,0,10,1,10,2,10,3,10,4,10,0,10,1,10,2,10,3,10,4,PBMC,0,50,100,150,200,250,FSC-H,0,50,100,150,200,250,SSC-H,CD20 PerCP-Cy5.5,CD3 PE,Zap70(Y319)/Syk(Y352)Alexa 647,10,0,10,1,10,2,10,3,10,4,0,20,40,60,80,100,10,0,10,1,10,2,10,3,10,4,0,20,40,60,80,100,10,0,10,1,10,2,10,3,10,4,0,20,40,60,80,100,10,0,101,10,2,10,3,10,4,10,0,10,1,10,2,10,3,10,4,CD3 PE,10,0,10,1,10,2,10,3,10,4,0,20,40,60,80,100,10,0,10,1,10,2,10,3,10,4,0,20,40,60,80,100,10,0,10,1,10,2,10,3,10,4,0,20,40,60,80,100,CD3-/CD20+,CD3+/CD20-,CD3-/CD20-,CD20 PerCP-Cy5.5,Whole Blood CD3 CrossLink,0,50,100,150,200,250,FSC-H,0,50,100,150,200,250,SSC-H,21.8,Multi-Color PhosFlow in CD3/CD28 or CD3 crosslinked human PBMCs or Whole Blood,CD3/CD28 Crosslink,Untreated cells(unshaded)vs treated cells(shaded),BD CBA,一种,“,三明治,”,免疫测定法,使用结合有高亲和性抗体的小球，用来特异性地捕获可溶性的抗原,.,用荧光检测抗体来检测被捕获的抗原,使用流式细胞仪进行检测分析,Multiplexed Beads,Shades of a color,Antibody coupled beads,emitting at distinct FL3 intensities,Various analytes,Antibody coupled PE label,emitting at FL2 intensity proportional to analyte conc.,Multiplexed Beads,0 pg/mL,80 pg/mL,1250 pg/mL,5000 pg/mL,FL3 Beads,FL2(PE),使用,BD Cytometric Bead Array(CBA),系统你可以,:,从单一小体积样本中得到多项检测结果,对所有的检测项目，只需要制备一个标准混合物来绘制标准曲线,避免人为造成的酶联放大的假阳性信号产生,用更少的时间和精力，却得到大量的结果,如果是用,488nm,和,635nm,两根激光器来实验，实验条件更容易建立,使用配有,HTS,选件的,BD,流式细胞仪可以做到自动平板上样和高通量检测,目前,CBA,主要提供的检测内容,可溶性细胞因子,细胞凋亡相关蛋白,磷酸化蛋白,BD CBA Flex Set,A,B,C,D,E,F,G,H,I,1,2,3,4,5,6,7,8,9,FL4-H or Red-A,FL3-H or NIR-A,Old Format BD CBA Beads,BD CBA Flex Set Beads,更为灵活强大的,CBA Flex Set,最多可同时检测,72,个项目,可自由选择搭配检测项目,检测项目范围更广泛,需要,488nm,和,635nm,两种波长的激光器,A,B,C,D,E,F,G,H,I,1,2,3,4,5,6,7,8,9,FL4-H or Red-A,FL3-H or NIR-A,9,个指标同时检测活化,T,细胞,1,2,3,4,5,6,7,8,9,1.Itk (Y511),2.ERK(T202/Y204),3.JNK(T183/Y185),4.P38(T180/Y182),5.PLC,g,(Y783),6.ZAP70(Y319),7.LAT(Y171),8.c-Jun(S63),9.RSK(S380),Kinetics of Jurkat Cell Activation With Anti-CD3/CD28,1,10,100,FOLD INCREASE IN UNITS/ML,0,5,10,15,20,TIME(MINUTES),RSK(S380),Jun(S63),LAT(Y171),Itk(Y511),ZAP-70(Y319),PLC,g,(Y783),P38(T180/Y182),JNK(T183/Y185),ERK(T202/Y204),P-Specific Antibodies,Monitoring the T Cell Activation Pathway by CBAControl),Lck,ZAP-70,P,Itk,P,P,MAPKKK,P,IKK,P,NF-kB,Ras,Grb2,S,O,S,Rac,P,P,PLC,P,P,DAG,PKC,P,V,A,V,SLP-76,P,LAT,P,P,P,P,P,P,P,P,P,P,P,P,Ras,MEK,ERK,JNK,p38,MKK4,7,MKK3,6,MEKK1-4,Elk,Jun,ATF2,MEKK4,LAT,P,P,RSK,Cells were activated with anti-CD3/CD28 for 2 minutes.SDS was added to a final concentration of 1%and the material was placed in a boiling water bath for 5 minutes.,Monitoring the T Cell Activation Pathway by CBA(PD-98059),Jurkat cells were pre-incubated with 200,m,M PD-98059(MEK inhibitor)for 20 minutes before being activated activated with anti-CD3/CD28 for 2 minutes.SDS was added to a final concentration of 1%and the material was placed in a boiling water bath for 5 minutes.,Monitoring the T Cell Activation Pathway by CBA(PP2),Jurkat cells were pre-incubated with 10,m,M PP2(Src family kinase inhibitor)for 20 minutes before being activated activated with anti-CD3/CD28 for 2 minutes.SDS was added to a final concentration of 1%and the material was placed in a boiling water bath for 5 minutes.,FCM in Molecular Biology,流式检测分子表型,特异的核酸序列,SNP,端粒长度,荧光报告蛋白检测基因表达情况,Fluorescent Proteins,DsRed,ZsYellow,HcRed,AmCyan,端粒长度的检测,端粒的长度和端粒酶的活性是现在研究的重点。因为它们被发现和细胞复制以及一些疾病的变化相关。,可以通过,FISH,，用带有荧光的核酸探针标记上端粒序列，通过流式细胞仪检测端粒的长度,流式分选应用举例,自然杀伤细胞分选,基因转染细胞的分选,绿色萤光蛋白分析（,GFP,）：左图显示,Hela,细胞转染了减毒,Mengo,病毒,vM16,，其中含转染后,8,小时与病毒,L,肽链融合的,GFP,。直方图显示,59,细胞表达该肽链，并显示不同的表达水平。重叠的红线表示阴性对照。,AND,.,肿瘤细胞分选,特异性,T,淋巴细胞分选,白血病细胞分选,