流式细胞术

流式细胞术是一种综合应用光学、机械学、流体力学、电子计算机、细胞生物学、分子免疫学等学科技术,对高速流动的细胞或亚细胞进行快速定量测定和分析的方法。它一秒钟能分析几千个细胞,并同时测定细胞的多个参数,广泛应用于生物医学的许多领域,如测定细胞的特征(形态、膜电位等)和细胞内pH,细胞DNA、蛋白质含量、表面受体、Ca2+等。对生物工程学来说,了解细胞的这些参数尤为重要,因为它们能比用传统技术测得的数据更好地描述细胞群体。从流式细胞仪对细胞多种参数的测定及原理,到它在生物工程学中的应用等方面进行了介绍,并讨论了流式细胞术的局限性和面临的挑战。     

抗鳗弧菌独特型单克隆抗体的制备及鉴定

利用具有中和活性的抗鳗弧菌单克隆抗体 4A6作为免疫原 ,通过单克隆抗体技术制备出 7株分泌抗独特型单抗的杂交瘤细胞。以ELISA竞争抑制实验及诱导Ab3的功能实验证实 ,其中 4株属于Ab2 β,有可能用于疫苗生产。     

流式细胞术计数细菌的研究

目的探讨用流式细胞术进行细菌计数。方法检测被链球菌α-34抑止而死亡的化脓性链球菌32309-2数量。结果实验管MFI值明显比对照管强,实验管本身的MFI强度随着时间的增加而增强,而对照管则基本上变化不是很大;到24h后,对照管MFI已达1．01,而对照管还是维持在0．4左右,两者T检验P〈0．01。结论用PI染色,可用流式细胞术检测死亡细菌。结果可靠,用时短,极大地简化了实验步骤。     

利用流式细胞术检测线粒体PTP开放

用线粒体电位特异必 光探针Ｒｈｏｄａｍｉｎｅ１２３标记线粒休后,在改装ＦＡＣＳ４２０流式细胞仪上用Ｃ－３０数据集集和储存程序采集并存储数据,用Ｃ－３０分析程序分析得取反映线料体前散射（ＦＳＣ）、Ｒｈｏｄａｍｉｎｅ１２３荧光强度和侧散射（ＳＳＣ） ＦＳＣ,ＦＬ１和ＦＬ２合并图。结果显示,２００μｍｏｌ／ＬＣａ＾２＋处理２０分钟使线粒体同时发生ＦＳＣ增大、Ｒｈｏｄａｍｉｎｅ１２３荧光强度降低和ＳＳＣ增     

流式细胞术在乳酸菌自溶检测中的应用

【目的】使用流式细胞术(Flow Cytometric)建立一种新的检测方法,可快速筛选自溶度不同的乳酸菌菌株。【方法】菌悬液经20mmol/L的PI-PBS染液在4℃条件下避光染色30min,上流式细胞仪进行测定,检测器激发光波长488nm,检测波长630nm,每个样品收集1×105个细胞,联机使用CellQuest软件分析结果。【结果】阳性染色细胞数与细胞总数之比很好地反映菌液中自溶细胞与非自溶细胞的比例关系,整个检测过程耗时仅为1h左右。【结论】与传统检测方法比较,FCM测定结果稳定可靠,检测时间短,为乳酸菌的自溶特性研究及筛选自溶度不同的菌株用作商业发酵剂提供了便利条件。     

基于液氮研磨法的流式细胞术检测马铃薯倍性的研究

染色体组倍性鉴定是马铃薯种质资源评价的重要内容,流式细胞仪能够快速、准确地对细胞核DNA含量进行测定,从而广泛用于检测植物染色体组倍性。建立适于马铃薯倍性鉴定的高通量流式细胞术体系,对马铃薯育种工作提供依据。以20份马铃薯合作88孤雌诱导后代为材料,用液氮研磨法制备叶片细胞核悬液,并将其与传统刀片切碎法制备的细胞核悬液进行比较,对已知四倍体马铃薯合作88和二倍体马铃薯IVP101进行染色体倍性测定,结果发现这两种方法在倍性测定结果之间无明显差异,但是液氮研磨法操作简单、耗时少。基于液氮研磨法的流式细胞术可快速、准确检测其倍性。另外,在液氮研磨法中,对细胞核悬液染色时间的长短(从15 min到12 h)并不会影响倍性测定结果,从而方便研究人员在实际操作中灵活选择染色时间。     

流式细胞术在植物细胞学研究中的应用

随着细胞生物学研究从传统的定性描述发展到定量、群体的研究而形成了一门新兴的交叉学科——分析细胞学。它从定量的角度对细胞的各种形态参数与生物学特性、生化成分组成以及细胞功能等进行研究。流式细胞（FCM）分析是其主要研究手段之一。FCM是将样品细胞悬浮于液体中,在流动过程中一个一个地通过测量区进行高速测量。其特点是：快速,每秒可分析数千个细胞;统计精度高,在短时间内可获得大量的细胞信息;进行多参数相关测量;分辨率高,可检出细胞间差异的5％。目前,流式细胞术已经比较广泛地应用于动物细胞周期分析、膜脂流动性…     

基于流式细胞术快速检测人鼻黏膜组织淋巴细胞亚群的实验研究

目的：基于流式细胞术检测正常人的鼻黏膜组织中的淋巴细胞亚群(CD3+T、CD4+T、CD8+T、NK、CD19+B)的比例,初步探讨 鼻局部黏膜免疫功能的意义,为鼻局部黏膜免疫性疾病的研究提供更多的参考。方法：用鼻黏膜刮匙获取21 例正常人的鼻黏膜 组织,并抽取其外周血2 mL。采用流式细胞术分别检测其鼻黏膜和外周血中的淋巴细胞亚群(包括CD3+T、CD4+T、CD8+T、NK、 CD19+B)分别所占的比例。结果：鼻黏膜和外周血的淋巴细胞亚群存在很大差异,与外周血相比,CD3+T 比例增加（t=15.34,P<0. 0001),CD4+T 比例降低(t=5.952,P<0.0001)、CD8+T 比例增加(t=12.44,P<0.0001)、NK 比例降低(t=4.865,P<0.0001)、CD19+B 比例降 低(t=15.56,P<0.0001),CD4+T/CD8+T 降低。结论：流式细胞术可以用来检测鼻黏膜的淋巴细胞亚群,鼻黏膜的淋巴细胞亚群和外 周血的淋巴细胞亚群存在很大差异,这种差异体现鼻黏膜组织独特的局部黏膜免疫功能,本方法为变应性鼻炎的研究提供了新 的研究途径。     

流式细胞术在哺乳动物精液质量检测中的应用

精液检测的首要目标就是快速准确地确定精子的生育力。同时具备多种特性和功能完整的精子才能受精,因而只有同时客观地检测多个指标,才能更好地反映精子的生育力。精子检测的传统方法费时费力,检测精子数量少,指标单一,而且易受操作者的主观影响,不能准确地反映精子功能。流式细胞术(FCM)为精子功能研究提供了一种快速、客观、多指标、大通量的检测手段;利用FCM检测精子的质膜完整性、顶体状态、染色质结构、线粒体功能以及细胞凋亡等,可以得知精子功能的相关情况。随着新的荧光探针、染色方法的不断开发和改进,FCM为精液质量检测提供了一种新的检测平台,应用前景极其广阔。     

适体及其在流式细胞术检测中的应用

适体是一种本质为RNA或DNA的小分子,它们能以配基形式像抗体一样与靶蛋白呈高亲合力特异性结合,可在一定程度上取代抗体用于检测和治疗。就适体筛选的指数富集配基系统进化技术原理、适体的优越性及其适体在流式细胞术检测中的应用进行了综述。     

A monoclonal antibody against synaptic AChE: a useful tool for detecting apoptotic cells

The classical function of acetylcholinesterase (AChE) is to terminate synaptic transmission at cholinergic synapses by rapidly hydrolyzing the neurotransmitter acetylcholine (ACh). Non-classical functions of AChE involve accelerating the assembly of Abeta peptide into amyloid fibrils and participating in haematopoiesis and neurite growth. Although numerous antibodies have been raised against AChE, many researchers have questioned their reliability to identify the AChE in situ, especially with the regard to its non-classical roles. Researchers attended the Ninth International Meeting on Cholinesterase raised this question by showing different Western blot patterns of AChE detected by different Abs. Producing more effective and reliable Abs for measuring AChE in vivo or in situ has become an important issue in many scientific fields. In this paper, we introduce a monoclonal antibody raised against synaptic AChE that we identified by Western blot assays, immunofluorescent staining and immunoprecipitation of AChE, and mass spectrometry. Our results strongly demonstrate the specificity of our monoclonal antibody to recognize synaptic AChE; hence our antibody can be used as an effective tool to study the various functions of AChE. Since the apoptosis-related AChE was its synaptic form, our antibody can be used as a tool to detect apoptotic cells.     

A rapid method for evaluation of cell number and viability by flow cytometry

A simple, rapid and reliable method has been developed for assessing the number and viability of cells, as well as cell size, in suspension culture by the use of flow cytometry. Propidium iodide exclusion is used for viability determination and fluorescent beads serve as an internal standard for cell enumeration. The main advantages of this method are its ability to handle a large number of samples with a high degree of precision and its specificity in detecting viable cells quantitatively in a heterogeneous culture of living and dead cells and debris. The method shows only a fraction of the variation found in the haemacytometer/trypan blue counting method due to its very low operator dependence. CHO - Chinese hamster ovary; FCS - Foetal calf serum; FS - Forward scatter light; MTT - 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; NCS - newborn calf serum; PBS - Phosphate buffered saline; PI - Propidium iodide; SS - Side scatter light. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

抗CD52单克隆抗体ADCC转基因测活方法的建立

目的 建立转基因细胞法测定抗CD52单克隆抗体(单抗)的抗体依赖细胞介导的细胞毒性作用(antibody-dependentcell-mediatedcytotoxicity,ADCC)生物学活性检测方法。方法 ①以Ramos细胞系作为靶细胞,以Jurkat-hFcγRⅢa/FcεRIγ-NFAT转基因细胞系作为效应细胞,通过荧光素酶检测系统(Bright-GloTM luciferase Assay System)建立抗CD52单抗的ADCC生物学活性检测方法;②对靶细胞、量效范围、效靶比及诱导时间进行优化并进行方法学验证;③研究建立的方法应用于对不同抗CD52单抗的ADCC生物学活性检测。结果 建立抗CD52单抗的ADCC生物学活性检测方法,抗CD52单抗在该方法中存在量效关系,符合四参数方程:y=(A-D)/[1+(x/C)B]+D;经优化后确定抗体量效范围为起始质量浓度360μg/mL,4倍系列稀释9个稀释度;效靶比为3∶1,诱导时间为6.0h;该方法具有良好的专属性;4个不同稀释组回收率样品经3次测定,相对效价分别为(45.58±4.67)%、(71.61±9.45)%、(122.92±7.92)%和(149.94±14.58)%;对应的回收率分别为(91.16±9.34)%、(95.49±12.60)%、(98.34±6.34)%和(99.96±9.72)%,上述结果的变异系数(coefficientofvariation,CV)均小于15%;且该方法也适用于不同抗CD52单抗的ADCC效应评价。结论 利用转基因细胞法成功建立了抗CD52单抗ADCC生物学活性检测方法,该方法专属性强、准确性高、重复性好,可用于评价抗CD52单抗的ADCC生物学活性。     

Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody

The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.     

Comparison of the production of a human monoclonal antibody against HIV-1 by heterohybridoma cells and recombinant CHO cells: A flow cytometric study

The production of human monoclonal antibodies for therapeutic use is of increasing importance for treatment of viral infections such as AIDS. As human x mouse heterohybridomas rarely reach the growth rates and cell specific production rates of mouse hybridomas the transfection of standard cell lines, such as CHO or BHK, is a promising alternative. This has the additional advantage that the IgG subtype can be changed to suit the desired application. However, the use of a cell line that has not originally developed to produce antibodies, as lymphocytes and myeloma cells have, might have unrecognised drawbacks. This will be especially significant in the case of antibodies as each molecule consists of 4 chains linked by disulphide bonds which require specific intracellular factors to be properly folded and processed (Heavy chain binding protein, Protein Disulfide Isomerase a.o.). In this study we have therefore compared two cell lines: a human x mouse heterohybridoma producing IAM-2F5, a human IgG₃ antibody specific for HIV-1 with neutralising properties and a Chinese Hamster Ovary cell transfected with dihydrofolate reductase and with the heavy and light chain genes of IAM-2F5 modified to IgG₁. From each cell line three subclones were selected with low, medium and high specific production rates. Batch cultures were performed and the following cellular parameters analysed by flow cytometry; 1) total RNA content (translational activity); 2) total protein content; 3) cell cycle phase distribution; 4) concentration of light and heavy chains; 5) concentration of helper proteins such as BiP and PDI. The production rate of heterohybridoma cells was best reflected in the intracellular concentration of kappa chain, while the gamma chain concentration was comparable for all three subclones. In the CHO cells the gamma chain expression and thus gene copy number appeared to be the limiting factor. The GRP78/BiP concentration in CHO remained unchanged in spite of a 5-fold higher concentration of gamma chain in the high producing subclone. The PDI concentration in CHO cells was much lower compared to the heterohybridoma cells, irrespective of production rates.Abbreviations PDI protein disulfide isomerase - GRP78/BiP Glucose regulated protein; Heavy chain binding protein     

流式细胞技术在微生物检测领域的应用研究

研究应用流式细胞技术(flow cytometry method,FCM)进行快速微生物检测的方法。与传统微生物检测方法相比,FCM法更快速和准确。经Pearson相关系数分析表明,在一定浓度范围内,FCM检测法与标准平板检测法(SPC)具有极强线性相关性。经Q检验法分析,FCM检测法具有良好的重复性。由此可见,FCM法可成为一种替代传统微生物检测法的自动化仪器检测新技术。     

庆大霉素单克隆抗体的制备及试剂盒的配制

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。     

鼠抗衣原体属抗原单克隆抗体的研制

本文利用杂交瘤技术,成功地建立了３株稳定分泌小鼠抗衣原体属脂多糖抗原单克隆抗体的杂交瘤细胞。实验结果表明,３株单抗均为ＩｇＭ类,特异性强,识别相似抗原位点,为今后的应用打下了基础。     

A new method for fish leucocyte counting and partial differentiation by flow cytometry

A simple and rapid method for analysis of fish blood cells is presented. Carp (Cyprinus carpio) blood was diluted 200 times with Hanks' solution containing 1 microg/ml of DiOC6(3) which is a fluorescent, lipophilic dye. After staining for 10 min, the blood cells were measured by a flow cytometer (FACS). Several blood cell populations were identified by different FL-1 (green fluorescence), FSC (forward scatter), and SSC (side scatter) properties. FL-1 v. SSC or FSC v. SSC dot-plot of stained blood cells displayed five separate cell populations: erythrocytes: a mixture of thrombocytes plus lymphocytes; monocytes; neutrophils; and basophils. The number of each type of blood cell counted by the FACS was in good agreement with those counted microscopically.