Application of Flow Cytometry to Cell Biology

实验免疫学分支（EIB）流式细胞术核心设施目前 支持欧洲投资银行50多名研究人员的多个研究项目， 在癌症研究中心（CCR）的其他地方。这些调查涉及 多参数定量单细胞分析和基于电子细胞分离 根据与从不同物种和/或组织新鲜制备的细胞相关的参数， 以及一系列体外培养细胞。提供基础研究支持， EIB的成员和NCI癌症研究中心的其他研究人员。 目前获得资助的项目包括但不限于以下研究领域： 通过细胞表面分子的细胞内信号传导的体内和体外分析; B） 分析动物和/或细胞中的细胞过程和/或缺陷， 修改; c）免疫发病机制和后果的研究; d） 分析多种分子的配位细胞表面表达; e） T细胞库产生的研究; g）移植物表达的分析 抗原; h）研究涉及T细胞谱系发育的机制; i） 细胞死亡机制;和j）干细胞分析。以下EIB/NCI/CCR项目是 由核心支持：PI：Alfred Singer BC 011106; T细胞功能在 发育中的胸腺细胞和T细胞中的细胞因子信号传导BC 011113; T 细胞存活BC 011114 ; microRNA在T细胞发育中的作用BC 009273; T细胞 分化和谱系选择BC 011116; MHC非依赖性T细胞BC 011117; T细胞 细胞因子信号传导的受体调节BC 011112;调节性T细胞的发育和功能 细胞PI：Richard Hodes BC 009265; T细胞库分析BC 009281;受体 介导的T和B细胞活化BC 009405;淋巴细胞增殖和 复制能力PI：Andre Nussenzweig BC 010283; DNA修复BC 010959;关系 DNA损伤检测和信号传导之间的关系BC 010961; DNA链断裂修复PI：Stephen Shaw 009257细胞免疫应答的机制 010272：促进人类蛋白质及其磷酸化信息的获取010993： 淋巴细胞皮质010994的分子结构：淋巴细胞膜近端嗜碱性 激酶010995：淋巴细胞中的ERM磷酸化010996：ERM的分子机制 调节PI; Dinah Singer BC 009285; MHC I类基因对外源性刺激的反应SC TAF 7：转录起始中的检查点调节因子BC 010927;染色质的作用 调控MHC Ⅰ类分子表达的结构BC 10928;边界元件调控MHC Ⅰ类分子 I在体内的表达BC 009279; MHC I类基因表达的调节PI：大卫西格尔 Toll样受体在获得性免疫产生中的作用 Toll样受体的结构和功能PI：Paul Roche BC 009404; MHC的调节 抗原呈递细胞BC 011033中的II类运输; MHC II类的机制 与质膜微区的结合BC 011035;免疫细胞胞吐的调节 细胞P1：Triantafyllos Chavakis BC 010663; JAM-C在细胞粘附相互作用中的作用 免疫细胞BC 010664;炎症和血管生成之间的串扰BC 010790;调节 PI：Hyun Park Z1A BC 011214; 白细胞介素-7受体表达的转录后调节Z1 A BC 011215;免疫 细胞因子信号转导抑制因子（SOCS）分子的调节作用 在此期间，核心研究中心还向以下非欧洲投资银行主要研究者提供了有限的研究支持：托马斯 Waldmann（代谢分支，CCR），Jay Berzofsky（疫苗分支，CCR），Genomiffa Franchini （疫苗分支，CCR）和保罗爱（国家儿童健康和发展研究所）。的 设施操作和维护2台操作员运行的3激光流式细胞仪，并进行细胞分选 和4台仅具有分析功能的用户操作流式细胞仪。该设施 最近获得了最先进的5激光，分析，流式细胞仪，将 用于扩展和扩展可用的应用程序。设施工作人员提供咨询， 研究人员在以下领域：实验设计，解决问题，试剂选择和 数据分析和解释。该设备支持多种流式细胞术 应用包括：罕见事件分析（包括干细胞分析）和细胞分选; 多色表型分析、细胞周期分析、增殖分析、代谢 分析包括钙通量分析、无菌细胞分选和细胞内细胞因子 分析。作为一项节约成本的措施，该设施还拥有一个超过150个的试剂库， 由机构人员预先滴定和等分的常用流式细胞术试剂 供多名EIB调查员使用。试剂库通过批量购买来降低成本， 最小化表征单个批次试剂所涉及的劳动和努力。的 一家工厂正在开发基于WINDOWS的流式细胞仪分析PC软件，该软件将提供 仪器制造商提供的软件中目前没有的功能，或 第三方软件来源。

The Experimental Immunology Branch (EIB) Flow Cytometry Core Facility currently supports multiple research projects for more than 50 investigators from within the EIB and elsewhere in the Center for Cancer Research (CCR). These investigations involve multiparametric quantitative single cell analysis of, and electronic cell separation based upon, parameters associated with cells freshly prepared from different species and/or tissues, as well as a spectrum of in vitro cultured cells. Basic research support is provided to members of the EIB and to other investigators within the Center for Cancer Research, NCI. Currently supported projects include, but are not limited to, the following areas of study: a) in vivo and in vitro analyses of intra-cellular signaling via cell surface molecules; b) analyses of cellular processes and/or defects in animals and/or cells with genetic modifications; c) studies of the mechanisms and consequences of immune pathogenesis; d) analyses of the coordinate cell surface expression of a variety of molecules; e) investigations of T cell repertoire generation; g) analyses of expression of transplantation antigens; h) investigations of mechanisms involved in T cell lineage development; i) mechanisms of cell death; and j) stem cell analyses. The following EIB/NCI/CCR Projects are supported by the core: PI: Alfred Singer BC 011106; Specification of T cell function during development BC 011111; Cytokine signaling in developing thymocytes and T cells BC 011113; T cell Survival BC 011114 ; Role of microRNAs in T cell development BC 009273; T Cell Differentiation and Repertoire Selection BC 011116; MHC-independent T cells BC 011117; T cell receptor regulation of cytokine signaling BC 011112; Development and function of regulatory T cells PI: Richard Hodes BC 009265; Analysis of the T Cell repertoire BC 009281; Receptor Mediated T and B Cell Activation BC 009405; Regulation of Lymphocyte Proliferation and Replicative Capacity PI: Andre Nussenzweig BC 010283; Dna repair BC 010959; Relationship between dna damage detection and signaling BC 010961;High-throughput screen for activators of DNA strand break repair PI: Stephen Shaw 009257 Mechanisms of Cellular Immune Responses 010272:Facilitating Access to Information on Human Proteins and Their Phosphorylation 010993: Molecular architecture of lymphocyte cortex 010994: Lymphocyte membrane-proximal basophilic kinases 010995: ERM phosphorylation in lymphocytes 010996: Molecular mechanisms of ERM regulation PI; Dinah Singer BC 009285; Responses of MHC Class I Genes to Exogeneous Stimuli SC 010375; TAF7: A Check-point Regulator in Transcription Initiation BC 010927; Role of Chromatin Structure in Regulating MHC Class I Expression BC 10928; Boundary Elements Regulate MHC Class I Expression in Vivo BC 009279; Regulation of Expression of MHC Class I Genes PI: David Segal BC 010879; Role of Toll-like receptors in the generation of acquired immunity BC 009254; Structure and Function of Toll-like Receptors PI: Paul Roche BC 009404; Regulation of MHC Class II Trafficking in Antigen Presenting Cells BC 011033; Mechanisms of MHC Class II Association with Plasma Membrane Microdomains BC 011035; Regulation of Exocytosis from Immune Cells PI: Triantafyllos Chavakis BC 010663; The Role of JAM-C in Adhesive Interactions of Immune Cells BC 010664; Crosstalk between inflammation and Angiogenesis BC 010790; Regulation of Leukocyte Integrins in Inflammatory Cell Recruitment PI: Hyun Park Z1A BC 011214; Post-Transcriptional Regulation of Interleukin-7 Receptor Expression Z1A BC 011215; Immune Regulatory Roles of Suppressor of Cytokine Signaling (SOCS) Molecules During this reporting period, the core also provided limited research support to the following non-EIB PI: Thomas Waldmann (Metabolism Branch, CCR), Jay Berzofsky (Vaccine Branch, CCR), Genoveffa Franchini (Vaccine Branch, CCR) and Paul Love (National Institute of Child Health and Development). The facility operates and maintains 2 operator run 3-laser flow cytometers with cell sorting capabilities and 4 user-operator flow cytometers with analysis only capabilities. The facility has recently acquired a state-of-the-art 5-laser, analysis only, flow cytometer that will be used to extend and expand available applications. Facility staff provide consultation to investigators in the areas of: experimental design, problem-solving, reagent selection and data analysis and interpretation. The facility supports a wide variety of flow cytometric applications including: rare event analysis (including stem cell analysis) and cell sorting; multi-color phenotypic analyses, cell cycle analysis, proliferation analysis, metabolic analyses including calcium flux analysis, sterile cell sorting, and intra-cellular cytokine analyses. The facility also, as a cost-savings measure, maintains a reagent bank of over 150 commonly used flow cytometry reagents that are pre-titred and aliquoted by facility personnel for use by multiple EIB investigators. The reagent bank minimizes costs by buying in bulk and minimizing labor and effort involved in characterizing individual batches of reagents. The facility is developing WINDOWS-based pc software for flow cytometry analysis that will provide capabilities not currently available in software available from instrument manufacturers or 3rd party software sources.