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来自EIB和癌症研究中心（CCR）其他地方的50多名研究人员的多个研究项目。这些调查涉及多参数定量单细胞分析，和电子细胞分离的基础上，从不同物种和/或组织新鲜制备的细胞，以及在体外培养的细胞谱相关的参数。基础研究支持提供给EIB的成员和NCI癌症研究中心的其他研究人员。目前资助的项目包括但不限于以下研究领域：a）通过细胞表面分子对细胞内信号进行体内和体外分析; B）分析动物和/或经遗传修饰的细胞的细胞过程和/或缺陷; c）研究免疫发病机制和后果; d）分析各种分子的协调细胞表面表达; e）研究T细胞库的产生; g）分析移植抗原的表达; h）研究涉及T细胞谱系发育的机制; i）细胞死亡的机制; j）干细胞分析和k）免疫基因调节的机制; l）通过转录因子、通过microRNA和通过细胞表面受体介导的活化途径控制分化和细胞功能的机制。以下EIB/NCI/CCR项目由核心支持：PI：Alfred Singer。BC 011106;开发期间T细胞功能的质量标准。BC 011111;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细胞分化和库选择。BC 011116; MHC非依赖性T细胞。BC 011117;细胞因子信号传导的T细胞受体调节。BC 011112;调节性T细胞的发育和功能。PI：Richard Hodes. BC 009265; T细胞库的分析。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的调节。PI：Dinah Singer. BC 009285; MHC I类基因对外源刺激的反应。BC 010375; TAF 7：转录起始中的检查点调节器。BC 009279; MHC I类基因表达的调节。BC 011381 BrD 4是一种调节转录的非典型激酶。BC 011425; Treg细胞中I类的表达。PI：Paul Roche. BC 009404;抗原呈递细胞中MHC II类运输的调节。BC 011033; MHC II类分子与质膜微区结合的机制。BC 011035;免疫细胞胞吐作用的调节。PI：Hyun Park. Z1 A BC 011214;白细胞介素-7受体表达的转录后调节。Z1 A BC 011215;细胞因子信号转导抑制因子（SOCS）分子的免疫调节作用。PI：Vanja Lazarevic。ZIA BC 011431 T-bet在实验性自身免疫性脑脊髓炎发病机制中的作用ZIA BC 011432通过microRNA调节TH 17细胞中的T-bet表达。PI：Damian Kovalovsky. ZIA BC 011429;淋巴细胞分化和功能中的Zbtb家族成员。在本报告所述期间，欧洲投资银行核心还向基因组完整性实验室提供了部分支助。主要研究者：Andre Nussenzweig，以及对以下非EIB PI的有限研究支持：托马斯瓦尔德曼（代谢分部，CCR）、托马斯里德（遗传学分支，CCR）、Paul Love（国家儿童健康与发育研究所）和Jeffrey Kopp（肾脏疾病分支，NIDDK）。该机构运行并维护两台操作员运行的多激光流式细胞仪，具有细胞分选功能，包括一台最先进的6激光细胞分选仪和5台仅具有分析功能的用户/操作员流式细胞仪，包括两（2）台最先进的5激光流式细胞仪分析仪。机构工作人员在以下领域为研究人员提供咨询：实验设计、问题解决、试剂选择和数据分析与解释。该设施支持各种流式细胞术应用，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，该机构还维护了一个包含150多种常用流式细胞仪试剂的试剂库，这些试剂由机构工作人员预先滴定和等分，供多名EIB研究者使用。试剂库通过批量购买和最小化表征单个批次试剂所涉及的劳动力和努力来最大限度地降低成本。该机构正在开发用于流式细胞术分析的基于WINDOWS的PC软件，该软件将提供仪器制造商或第三方软件源提供的软件目前无法提供的功能。

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; j) stem cell analyses and k) mechanisms of immune gene regulation; l) mechanisms of control of differentiation and cell function by transcription factors, by microRNA, and by cell surface receptor-mediated activation pathways. 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: Dinah Singer. BC 009285; Responses of MHC Class I Genes to Exogeneous Stimuli. BC 010375; TAF7: A Check-point Regulator in Transcription Initiation. BC 009279; Regulation of Expression of MHC Class I Genes. BC 011381 BrD4 is an atypical kinase that regulates transcription. BC 011425; Expression of class I in Treg cells. 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: 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. PI: Vanja Lazarevic. ZIA BC 011431 Role of T-bet in the pathogenesis of experimental autoimmune encephalomyelitis. ZIA BC 011432 Regulation of T-bet expression in TH17 cells by microRNAs. PI: Damian Kovalovsky. ZIA BC 011429; Zbtb family members in lymphocyte differentiation and function. During this reporting period, the EIB core also provided partial support to the Laboratory of Genome Integrity. PI: Andre Nussenzweig, and limited research support to the following non-EIB PI: Thomas Waldmann (Metabolism Branch, CCR),Thomas Ried, (Genetics Branch, CCR), Paul Love (National Institute of Child Health and Development, and Jeffrey Kopp (Kidney Diseases Branch, NIDDK). The facility operates and maintains two operator-run multi-laser flow cytometers with cell sorting capabilities including a state-of-the-art 6-laser cell sorter and 5 user/operator flow cytometers with analysis only capabilities including two(2) state-of-the-art 5-laser flow cytometer analyzers. 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.