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和其他癌症研究中心（CCR）的50多名研究人员的多个研究项目。这些研究涉及多参数定量单细胞分析和基于不同物种和/或组织新鲜制备的细胞相关参数的电子细胞分离，以及体外培养细胞的光谱。基础研究支持提供给EIB成员和NCI癌症研究中心的其他研究人员。目前支持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的体内和体外分析；B)动物和/或基因修饰细胞的细胞过程和/或缺陷分析；C)免疫发病机制和后果的研究；D)各种分子的坐标细胞表面表达分析；e) T细胞库生成的研究；G)移植抗原表达分析；h)研究涉及T细胞谱系发育的机制；1)细胞死亡机制；J)干细胞分析和k)免疫基因调控机制；l)通过转录因子、microRNA和细胞表面受体介导的激活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。公元前011106年;T细胞发育过程中的功能说明。公元前011111年;发育中的胸腺细胞和T细胞的细胞因子信号传导。公元前011113年;T细胞存活。BC 011114；microrna在T细胞发育中的作用。公元前009273年;T细胞分化和细胞库选择。公元前011116年;mhc非依赖性T细胞。公元前011117年;T细胞受体对细胞因子信号的调控。公元前011112年;调节性T细胞的发育和功能。PI：理查德·霍兹。公元前009265年;T细胞库分析。公元前009281年;受体介导的T细胞和B细胞活化。公元前009405年;淋巴细胞增殖和复制能力的调控。PI：黛娜·辛格。公元前009285年;MHC I类基因对外源刺激的反应。公元前010375年;TAF7：转录起始的检查点调节因子。公元前009279年;MHC I类基因的表达调控。BrD4是一种调节转录的非典型激酶。公元前011425年;I类在Treg细胞中的表达。PI：保罗·罗奇。公元前009404年;抗原呈递细胞中MHC II类运输的调控。公元前011033年;MHC II类与质膜微结构域相关的机制。公元前011035年;免疫细胞胞吐的调节。PI：朴铉。Z1a BC 011214；白介素-7受体表达的转录后调控。Z1a BC 011215；细胞因子信号抑制因子（SOCS）分子的免疫调节作用PI: Vanja Lazarevic。T-bet在实验性自身免疫性脑脊髓炎发病机制中的作用微rna对TH17细胞T-bet表达的调控。PI: Damian Kovalovsky。齐亚BC 011429；Zbtb家族成员在淋巴细胞分化和功能中的作用。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持。PI: Andre Nussenzweig，并为以下非eib PI提供有限的研究支持：Thomas Waldmann（代谢分支，CCR），Thomas Ried（遗传学分支，CCR）， Paul Love（国家儿童健康与发展研究所）和Jeffrey Kopp（肾脏病分支，NIDDK）。该设施运营和维护两台具有细胞分选能力的操作员运行多激光流式细胞仪，包括一台最先进的6激光细胞分选仪和5台仅具有分析能力的用户/操作员流式细胞仪，包括两台最先进的5激光流式细胞仪分析仪。设施工作人员在实验设计、问题解决、试剂选择和数据分析和解释等方面为调查人员提供咨询。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。作为一项节省成本的措施，该设施还维护了一个包含150多种常用流式细胞仪试剂的试剂库，这些试剂由设施人员预滴定和引用，供多个EIB研究人员使用。试剂库通过批量购买和最大限度地减少人工和努力表征单个批次试剂的成本。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.