多色荧光原位杂交检测人卵细胞染色体非整倍体方法的建立

目的 建立多色荧光原位杂交技术检测人卵细胞染色体非整倍体的方法。方法 取试管婴儿助孕技术后未能受精成功的卵细胞，于取卵后13d固定，采用多色荧光原位杂交方法检测卵细胞13，16，18。21和22号染色体的情况。结果 正常未受精卵细胞中期染色体显示一个成对的杂交信号，每条染色单体显示一个单个信号；分裂相中多出或缺少一个成对杂交信号表明多余或缺少一条染色体；分裂相中多出或缺少一个单个信号表明多余或缺少一条染色单体；两个单个信号分离表明两条姐妹染色单体分离。结论 采用多色荧光原位杂交方法可以有效检测人卵细胞染色体非整倍体异常。     

FCM检测标记细胞凋亡方法的建立及应用

采用流式细胞仪 (FCM )测定BXSB狼疮鼠发病组、正常对照组和中药治疗组的脾脏组织中CD4 + 、CD19+细胞凋亡的水平 ,结果显示 ,BXSB狼疮鼠发病组CD4 + 、CD19+ 细胞凋亡水平显著高于正常对照组和中药治疗组(P <0 .0 5 )。此法需样本量少 ,操作简便 ,结果可靠 ,可在临床及科研中推广使用     

流式细胞术检测细胞毒方法的建立

目的：建立一种用流式细胞仪测定细胞介导细胞毒活性的方法。方法：用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记靶细胞，再用碘化丙碇(PI)标记DNA筛选细胞膜已破坏的靶细胞。效靶比为100：1—6．25：1，共孵育时间为1-6小时，流式细胞仪收集1000个靶细胞并测定被杀细胞的百分率。结果：CFSE是合适的标记靶细胞的荧光染料，18小时后也能很好地区分效靶细胞；靶细胞的自然死亡率低于5％；杀伤百分率随着效靶比和共孵育时间的增加而增加。最佳效靶比为50：1—25：1，共孵育时间为2—4小时。结论：CFSE／PI双荧光染料标记的流式细胞分析检测细胞毒具有多个优点：避免放射性试剂的应用，敏感性增强，单细胞水平的分析。     

人外周血内皮祖细胞培养方法的建立

背景：成人外周血来源丰富,但内皮祖细胞含量较少,为使其能够更好的应用于组织工程及细胞治疗,有必要建立外周血内皮祖细胞成熟、稳定的体外扩增体系。 目的：建立稳定的人外周血分离、培养和体外扩增血管内皮祖细胞的方法。 方法：应用密度梯度离心法,获取外周血单个核细胞,将分选后细胞接种于预先包埋了人纤维连接蛋白的培养板上,加入内皮祖细胞专用培养基中培养3 d后,洗掉非贴壁细胞,培养至第6天,收集贴壁细胞,应用倒置显微镜和苏木精-伊红染色进行细胞形态学观察;采用MTT法和细胞计数测定第1,3代细胞生长曲线;应用流式细胞仪测定祖细胞和内皮细胞系标志,对培养的细胞进行鉴定。 结果与结论：细胞生长曲线测定表明接种后第3天细胞进入指数增生期,至第6天进入平台期,随着传代次数的增加,细胞增殖速度变慢,同时表达干细胞表面标志CD34、CD133和内皮细胞表面标志血管性血友病因子、血管内皮生长因子受体2。证明人外周血可以分离培养内皮祖细胞。     

多色流式细胞术对40例急性白血病免疫分型的研究

白血病的形态学、免疫学、细胞遗传学和分子生物学分型已成为现代白血病诊断所必需,其中流式细胞术免疫分型已成为急性白血病(AL)诊断和分型的重要依据,具有重要的临床诊断意义.我们采用流式细胞仪四色荧光标记技术,CD45/SSC双参数散点图设门方法,可全面地与特异地检测出白血病细胞抗原的分布,使白血病免疫分型的结果更为准确和客观[1].     

流式细胞免疫分型在B细胞淋巴瘤诊断中的应用

随着流式细胞术及单克隆抗体技术的发展，流式细胞术以其快捷、敏感并能同时对多个参数进行定量分析等优点被广泛应用于淋巴造血系统疾病诊断及鉴别诊断中。目前在我国部分医院，     

流式荧光微球技术的应用现状及进展

流式荧光微球技术是一种新的高通量检测技术,该技术是以荧光编码微球作为传统免疫学测定、亲和力测定及DNA杂交测定的固相载体,通过流式细胞仪进行检测的新的可用于高通量筛查的多路测定法.通过使用不同荧光编码的微米大小的聚合微球,流式荧光微球技术可以同时分析复杂样品中的多种分析物.每一种微球表面被修饰使其与相应的待测抗原、抗体或寡核苷酸反应,将不同微球混合即可进行多重复合测定.该技术结合了荧光编码微球的特异性与流式细胞仪的高度敏感性.作为一个技术分析平台,流式荧光微球技术在科研及临床应用中将具有重要的潜力.就其应用现状及进展作一综述.     

卵巢上皮性肿瘤细胞DNA含量流式细胞仪分析

卵巢上皮性肿瘤细胞ＤＮＡ含量流式细胞仪分析傅兴生，徐燕兰，魏宝秀，潘凌云我们采用流式细胞仪分析４４例用石蜡包埋的卵巢上皮性肿瘤的细胞核ＤＮＡ含量、细胞周期各时相分布与临床关系。一、材料与方法我们选择１９７２～１９８８年之间的４８例卵巢上皮性肿瘤、其中...     

基于流式细胞术的scFv抗体库筛选技术的建立

目的 利用NlpA前导肽诱导抗体锚定细菌内膜建立筛选scFv抗体库的展示技术,为高亲和力抗体的筛选奠定基础.方法 从pNAD质粒中克隆出NlpA前导肽基因序列,酶切后将该序列克隆进pHEN1表达载体中.以PEAI质粒为模板,利用PCR的方法克隆得到抗-hIL-1β抗体的重链可变区和轻链可变区基因,然后利用重叠PCR的方法构建得到抗-hIL-1β scFv抗体.将scFv抗体插入到NlpA leader-pHEN1表达载体中构建成展示scFv的重组质粒pBSD-scFv.将pBSD-scFv转入到E.coli DH5α中诱导表达,原生质球制备后,采用梯度浓度的抗原进行孵育,最后经流式细胞术(FCM)检测抗体展示情况并且分选阳性群体,利用质粒提取的方法来替代PCR方法拯救阳性基因,转化E.coli DH5α,利用FCM再次检测该群体展示的抗体与抗原结合情况.结果 所展示的抗-hIL-1β scFv抗体依次与抗原和FITC标记的抗原特异性抗体孵育后,用FCM实时检测,结果显示出很强的荧光信号并且表现出抗原浓度依赖性.拯救出的pBSD-scFv-原生质球的FCM检测结果与首次展示的FCM结果一致,该系统能够稳定的展示抗体.结论 经过该细菌展示系统展示的scFv抗体能够有效的折叠,与相应的抗原具有很好的特异结合能力.     

Quantification of mucosal mononuclear cells in tissues with a fluorescent bead-based polychromatic flow cytometry assay

Since the vast majority of infections occur at mucosal surfaces, accurate characterization of mucosal immune cells is critically important for understanding transmission and control of infectious diseases. Standard flow cytometric analysis of cells obtained from mucosal tissues can provide valuable information on the phenotype of mucosal leukocytes and their relative abundance, but does not provide absolute cell counts of mucosal cell populations. We developed a bead-based flow cytometry assay to determine the absolute numbers of multiple mononuclear cell types in colorectal biopsies of rhesus macaques. Using 10-color flow cytometry panels and pan-fluorescent beads, cells were enumerated in biopsy specimens by adding a constant ratio of beads per mg of tissue and then calculating cell numbers/mg of tissue based on cell-to-bead ratios determined at the time of sample acquisition. Testing in duplicate specimens showed the assay to be highly reproducible (Spearman R=0.9476, P<0.0001). Using this assay, we report enumeration of total CD45(+) leukocytes, CD4(+) and CD8(+) T cells, B cells, NK cells, CD14(+) monocytes, and myeloid and plasmacytoid dendritic cells in colorectal biopsies, with cell numbers in normal rhesus macaques varying from medians of 4486 cells/mg (T cells) to 3 cells/mg (plasmacytoid dendritic cells). This assay represents a significant advancement in rapid, accurate quantification of mononuclear cell populations in mucosal tissues and could be applied to provide absolute counts of a variety of different cell populations in diverse tissues.     

Rapid yeast DNA staining method for flow cytometry

The variation of the fluorescence intensity of olivomycin-stained yeast cells as a function of the concentration of olivomycin, NaCl, and MgCl2 in the staining solution was studied. The best results were obtained when the staining solution contained 100 micrograms, olivomycin/ml, 40 mM MgCl2, and 1 M NaCl. A staining time of 12 min was sufficient for proper staining.     

Exhaustive expansion: A novel technique for analyzing complex data generated by higher-order polychromatic flow cytometry experiments

Background  

The complex data sets generated by higher-order polychromatic flow cytometry experiments are a challenge to analyze. Here we describe Exhaustive Expansion, a data analysis approach for deriving hundreds to thousands of cell phenotypes from raw data, and for interrogating these phenotypes to identify populations of biological interest given the experimental context.     

Determination of mixed chimerism by a simple flow cytometry method

A simple, sensitive and accurate method was developed to determine the level of lymphoid chimerism in bone marrow-transplanted rodents. The method is based on flow cytometry using polyclonal alloantisera and labeled second step anti-IgG antibodies. Using mixtures of spleen cells from different mouse strains, it was demonstrated that low levels of chimeric cells (less than 1%) could easily be detected. Moreover, using two-color fluorescence analysis, the level of chimerism could also be determined in subpopulations of lymphoid cells, e.g., CD4 or CD8 cells and was found to be identical to the results obtained in unseparated lymphoid populations. The method was compared to the complement dependent cytotoxicity assay (CDCA) and to the flow cytometric determination of chimerism using labeled monoclonal antibodies against specific MHC antigens. CDCA was found to be more labor intensive and could only estimate the composition of the cell mixtures without detecting low levels of chimerism (< 5%). The results of flow cytometry, using directly labeled monoclonal antibodies or polyclonal antibodies with second step reagents, were identical.

It is concluded that, due to its simplicity and high sensitivity, the method described permits reliable determination of the level of mixed chimerism in rodents and is an excellent alternative when no anti-MHC mAbs are available.     

A general method for bead-enhanced quantitation by flow cytometry

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry.     

流式细胞术检测淋巴细胞增殖方法的建立

目的 :建立一种用流式细胞仪同时测定T淋巴细胞增殖与细胞因子分泌的方法。方法 :用非特异性刺激剂PMA、PHA刺激 1 0例正常人外周血单个核细胞 ,体外培养 2、4、6、8、1 0h ,流式细胞术在单细胞水平检测T淋巴细胞的功能性激活、表面活化标志性抗原 (CD69)的表达、DNA合成 (BrdU掺入法 )及胞内细胞因子 (IL 4、IFN γ)分泌。选择合适的刺激剂、调整细胞浓度、优化实验方法 ,确定最佳实验条件。结果 :BrdU掺入法测定T淋巴细胞增殖 ,最佳的反应细胞浓度为 1× 1 0 6ml-1 ,比较不同刺激剂和体外培养时间 :PMA(2 0ng ml)刺激后 8hT细胞中CD69+、BrdU+双阳性细胞比例最高 (82 3 %± 7 2 % ) ,IFN γ+、IL 4+细胞百分率分别为 2 5 2 %± 3 7%和 3 4%± 1 6 % ,均显著高于未刺激组 (P <0 0 1 )。结论 :应用流式细胞术进行单个核细胞的增殖分析可以同时检测细胞表面活化抗原的表达、DNA合成及胞内细胞因子的分泌。此方法能够分析不同亚群细胞对于不同刺激原的反应和激活表型。敏感性高、重复性好 ,可在单细胞水平检测单个核细胞的增殖和功能性激活     

Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.     

Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry.

Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.