Application of Flow Cytometry to Cell Biology

实验免疫学分部(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。这些研究包括对新鲜从不同物种和/或组织制备的细胞以及体外培养细胞的光谱进行多参数定量单细胞分析和基于与细胞相关的参数的电子细胞分离。基础研究支持提供给EIB成员和NCI癌症研究中心内的其他研究人员。目前得到支持的项目包括但不限于以下研究领域：a)通过细胞表面分子进行的细胞内信号传递的体内和体外分析；b)动物和/或基因修饰细胞的细胞过程和/或缺陷的分析；c)免疫发病机制和后果的研究；d)各种分子的细胞表面配位表达的分析；e)T细胞库的研究；g)移植抗原的表达分析；h)T细胞谱系发育的机制的研究；i)细胞死亡的机制；J)干细胞分析和k)免疫基因调节机制。以下EIB/NCI/CCR项目得到核心支持：PI：Alfred SingerBC 011106；发育过程中T细胞功能的规范BC 011111；胸腺细胞和T细胞发育中的细胞因子信号转导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非依赖的T细胞BC 011117；T细胞受体对细胞因子信号的调节BC 011112；调节性T细胞的发展和功能PI：Richard HodesBC 009265；T细胞库BC 900281；T和B细胞受体激活BC 009405；淋巴细胞增殖和复制能力的调节PI：Stephen Shaw009257细胞免疫反应的机制010272：促进人类蛋白质及其磷酸化信息的获取010993：淋巴细胞膜的分子结构：近端嗜碱性蛋白激酶010994：淋巴细胞膜-近端嗜碱性运动ERM磷酸化010996：ERM调节的分子机制PI；Dinah SingerBC 009285；MHC I类基因对外源刺激SC 010375的反应；TAF7：转录启动BC 010927中的检查点调节；染色质结构在调节BC 10928表达中的作用；边界元件调节ViBC中I类HC的表达；MHC I类基因表达的调节PI：David SegalBC 010879；Toll样受体在免疫获得性生成中的作用；900BC；900BC。Toll样受体的结构和功能PI：Paul RocheBC 009404；MHC Class II在抗原提呈细胞中运输的调节BC 011033；MHC Class II与质膜微区结合的机制BC 011035；免疫细胞胞吐的调节PI：Hyun ParkZ1ABC 011214；白细胞介素7受体表达的转录后调节PI：Z1ABC 011215；细胞因子信号抑制因子(SOCS)分子的免疫调节作用PI：Vanja Lazareviczia 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ZBC 011432微核糖核酸对T-bet表达的调节PI：Damian Kovalovsky ZBC 01429；Zbtb家族成员在淋巴细胞分化和功能中的作用PI：Andre NuszwezBC 0283；DNA修复BC 010959；DNA损伤检测和信号BC 010961之间的关系；DNA链断裂修复激活子的高通量筛查在本报告所述期间，CORE还为以下非EIB PI提供了有限的研究支持：Thomas Waldmann(CCR新陈代谢分会)，Jay Berzofsky(疫苗分会)，凯瑟琳·凯利(CCR细胞和癌症生物学分会)和Paul Love(国家儿童健康与发育研究所)。在该实验室成立后，该中心还继续为Andre Nussenzweig(基因组完整性实验室，CCR)提供有限的支持。该实验室运营和维护两台操作员运行的具有细胞分选功能的多激光流式细胞仪，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，其中包括两(2)最先进的5激光流式细胞仪分析仪。设施工作人员为研究人员提供以下方面的咨询：实验设计、解决问题、试剂选择以及数据分析和解释。该设施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)和细胞分类；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)、无菌细胞分类和细胞内细胞因子分析。作为节省成本的措施，该设施还保留了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先命名和等量，供多名EIB调查人员使用。试剂库通过批量购买并最大限度地减少表征单个批次试剂所涉及的人力和精力来最大限度地降低成本。该设施正在开发基于Windows的PC软件，用于流式细胞仪分析，该软件将提供目前仪器制造商或第三方软件来源提供的软件所不具备的功能。

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.The following EIB/NCI/CCR Projects are supported by the core:PI: Alfred SingerBC 011106; Specification of T cell function during developmentBC 011111; Cytokine signaling in developing thymocytes and T cellsBC 011113; T cell SurvivalBC 011114 ; Role of microRNAs in T cell developmentBC 009273; T Cell Differentiation and Repertoire SelectionBC 011116; MHC-independent T cellsBC 011117; T cell receptor regulation of cytokine signalingBC 011112; Development and function of regulatory T cellsPI: Richard HodesBC 009265; Analysis of the T Cell repertoireBC 009281; Receptor Mediated T and B Cell ActivationBC 009405; Regulation of Lymphocyte Proliferation and Replicative CapacityPI: Stephen Shaw009257 Mechanisms of Cellular Immune Responses010272:Facilitating Access to Information on Human Proteins and Their Phosphorylation010993: Molecular architecture of lymphocyte cortex010994: Lymphocyte membrane-proximal basophilic kinases010995: ERM phosphorylation in lymphocytes010996: Molecular mechanisms of ERM regulationPI; Dinah SingerBC 009285; Responses of MHC Class I Genes to Exogeneous StimuliSC 010375; TAF7: A Check-point Regulator in Transcription InitiationBC 010927; Role of Chromatin Structure in Regulating MHC Class I ExpressionBC 10928; Boundary Elements Regulate MHC Class I Expression in VivoBC 009279; Regulation of Expression of MHC Class I GenesPI: David SegalBC 010879; Role of Toll-like receptors in the generation of acquired immunityBC 009254; Structure and Function of Toll-like ReceptorsPI: Paul RocheBC 009404; Regulation of MHC Class II Trafficking in Antigen Presenting CellsBC 011033; Mechanisms of MHC Class II Association with Plasma Membrane MicrodomainsBC 011035; Regulation of Exocytosis from Immune CellsPI: Hyun ParkZ1A BC 011214; Post-Transcriptional Regulation of Interleukin-7 Receptor ExpressionZ1A BC 011215; Immune Regulatory Roles of Suppressor of Cytokine Signaling (SOCS) MoleculesPI: Vanja LazarevicZIA BC 011431 Role of T-bet in the pathogenesis of experimental autoimmune encephalomyelitisZIA BC 011432 Regulation of T-bet expression in TH17 cells by microRNAs PI: Damian KovalovskyZIA BC 011429; Zbtb family members in lymphocyte differentiation and functionPI: 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 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), Kathleen Kelly (Cell and Cancer Biology Branch, CCR) and Paul Love (National Institute of Child Health and Development). The core has also continued to provide limited support to Andre Nussenzweig (Laboratory of Genome Integrity, CCR) subsequent to establishment of that laboratory.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.