Application of Flow Cytometry to Cell Biology

流式细胞术在细胞生物学中的应用

• 批准号: 10262723
• 负责人: SUSAN SHARROW
• 金额: $ 198.06万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262723
• 关键词: Animals; Antigen-Presenting Cells; Area; Award; B-Cell Activation; Basic Science; Biology; CISH gene; Cancer Research Project; Cell Cycle; Cell Death; Cell Lineage; Cell Separation; Cell Survival; Cell membrane; Cell physiology; Cell surface; Cells; Cellular biology; Color; Computer software; Consultations; Core Facility; Cost Savings; Cultured Cells; Cytokine Signaling; Data; Data Analyses; Defect; Dendritic Cells; Development; Disease; Epithelial Cells; Event; Experimental Autoimmune Encephalomyelitis; Experimental Designs; FOXO1A gene; Factor Analysis; Flow Cytometry; Funding; Gene Expression Regulation; Generations; Genetic; Genetic Transcription; Goals; Histocompatibility Antigens; Human Resources; Immune; Immune system; Immunobiology; Immunology; In Vitro; Individual; Interleukin 7 Receptor; Investigation; Lasers; Lymphoid Cell; MHC Class I Genes; MHC Class II Genes; Manufacturer Name; Measures; Mediating; Membrane Microdomains; Metabolic; MicroRNAs; Modification; Mucosal Immune Responses; Multiparametric Analysis; NCI Center for Cancer Research; National Cancer Institute; Pathogenesis; Phenotype; Phosphotransferases; Post-Transcriptional Regulation; Problem Solving; Reagent; Regulation; Regulation of Exocytosis; Regulatory T-Lymphocyte; Research Personnel; Research Project Grants; Research Support; Role; Running; Signal Transduction; Signaling Molecule; Source; Sterility; Stimulus; T cell differentiation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; TAF7 gene; Techniques; Technology; Thymic epithelial cell; Thymus Gland; Tissues; Transcription Initiation; base; cost; cytokine; in vivo; instrument; instrumentation; intestinal barrier; intestinal epithelium; intraepithelial; laser cell sorter; lymphocyte proliferation; member; receptor; receptor expression; release of sequestered calcium ion into cytoplasm; response; single cell analysis; stem cells; thymocyte; trafficking; transcription factor

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 multi parametric quantitative single cell analysis of, and electronic cell separation of cells, 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 Singer; ZIA BC 009273 T Cell Differentiation and Repertoire Selection, ZIA BC 011106 Specification of T cell function during development, Z1A BC 011111 Cytokine signaling in developing thymocytes and T cells, ZIA BC 011112 Development and function of regulatory T cells, Z1A BC 011113 T cell Survival, Z1A BC 011114 Role of microRNAs in T cell development, Z1A BC 011116 MHC-independent T cells, Z1A BC 011117 T cell receptor regulation of cytokine signaling. PI: Richard Hodes: Z1A BC 009265; Analysis of the T Cell repertoire, Z1A BC 009281 Receptor Mediated T and B Cell Activation, Z1A BC 009405; Regulation of Lymphocyte Proliferation and Replicative Capacity. PI: Dinah Singer Z1A BC 009285 Responses of MHC Class I Genes to Exogeneous Stimuli, Z1A SC 010375 TAF7: A Check-point Regulator in Transcription Initiation, Z1A BC 009279 Regulation of Expression of MHC Class I Genes, ZIA BC 011381 Brd4 is an atypical kinase that regulates transcription, ZIA BC 011425 Expression of class I in Treg cells. PI: Paul Roche Z1A BC 009404 Regulation of MHC Class II Trafficking in Antigen Presenting Cells, Z1A BC 011033 Mechanisms of MHC Class II Association with Plasma Membrane Microdomains, Z1A 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: Chuan Wu ZIA BC 011755 The role of Foxo1 for intestinal epithelial cells during mucosal immune response, ZIA BC 011801 CD101 controls intraepithelial cells (IELs) for intestinal barrier integrity. PI: Yousuke Takahama ZIA BC 011806 Thymus Biology. 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 members 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 but not limited to analysis/cell sorting of subpopulations of stem cells, thymic epithelial cells, innate lymphoid cells (ilc), T regulatory cells, and dendritic cells); analysis and cell sorting of a variety of immune system cells in studies of development, activation and response to disease. These various studies employ multi-color phenotypic analyses, cell cycle analysis, proliferation analysis, metabolic analyses including calcium flux analysis, sterile cell sorting, and intra-cellular cytokine and transcription factor 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 source. During the period, the facility competed for and was awarded funds to purchase 3 new state-of-the are flow cytometers. Two of these will replace existing, obsolete instrumentation and one additional instrument will increase capacity of the facility.

实验免疫学分支 (EIB) 流式细胞术核心设施目前支持来自 EIB 内部和癌症研究中心 (CCR) 其他地方的 50 多名研究人员的多个研究项目。这些研究涉及基于与从不同物种和/或组织新鲜制备的细胞以及一系列体外培养细胞相关的参数，对细胞进行多参数定量单细胞分析和细胞电子细胞分离。向 EIB 成员和 NCI 癌症研究中心的其他研究人员提供基础研究支持。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传导进行体内和体外分析； b) 对动物和/或基因修饰细胞的细胞过程和/或缺陷进行分析； c) 免疫发病机制和后果的研究； d) 多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) T细胞谱系发育相关机制的研究； i) 细胞死亡机制； j) 干细胞分析和 k) 免疫基因调控机制。核心支持以下 EIB/NCI/CCR 项目： PI：Alfred Singer； ZIA BC 009273 T 细胞分化和全集选择，ZIA BC 011106 发育过程中 T 细胞功能规范，Z1A BC 011111 发育中胸腺细胞和 T 细胞中的细胞因子信号传导，ZIA BC 011112 调节性 T 细胞的发育和功能，Z1A BC 011113 T 细胞存活，Z1A BC 011114 microRNA 在 T 细胞发育中的作用， Z1A BC 011116 MHC 独立 T 细胞，Z1A BC 011117 T 细胞受体对细胞因子信号传导的调节。 PI：理查德·霍兹：Z1A BC 009265； T 细胞库分析，Z1A BC 009281 受体介导的 T 和 B 细胞激活，Z1A BC 009405；淋巴细胞增殖和复制能力的调节。 PI：Dinah Singer Z1A BC 009285 MHC I 类基因对外源刺激的反应，Z1A SC 010375 TAF7：转录起始中的检查点调节因子，Z1A BC 009279 MHC I 类基因表达的调节，ZIA BC 011381 Brd4 是一种调节转录的非典型激酶，齐亚BC 011425 Treg 细胞中 I 类的表达。 PI：Paul Roche Z1A BC 009404 抗原呈递细胞中 MHC II 类运输的调节，Z1A BC 011033 MHC II 类与质膜微域关联的机制，Z1A BC 011035 免疫细胞胞吐作用的调节。 PI：Hyun Park Z1A BC 011214 IL-7 受体表达的转录后调节，Z1A BC 011215 细胞因子信号传导 (SOCS) 分子抑制剂的免疫调节作用。 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 microRNA 在 TH17 细胞中调节 T-bet 表达。 PI：Chuan Wu ZIA BC 011755 Foxo1 在粘膜免疫反应过程中对肠上皮细胞的作用，ZIA BC 011801 CD101 控制上皮内细胞 (IEL) 以维持肠道屏障的完整性。 PI：高滨洋介 ZIA BC 011806 胸腺生物学。该设施运营和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 台用户/操作员流式细胞仪，仅具有分析功能，包括两 (2) 台最先进的 5 激光流式细胞仪分析仪。设施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（包括但不限于干细胞亚群、胸腺上皮细胞、先天淋巴细胞 (ilc)、T 调节细胞和树突状细胞的分析/细胞分选）；在疾病的发育、激活和反应研究中对多种免疫系统细胞进行分析和细胞分选。这些不同的研究采用多色表型分析、细胞周期分析、增殖分析、代谢分析（包括钙流分析）、无菌细胞分选以及细胞内细胞因子和转录因子分析。作为一项节省成本的措施，该机构还维护着一个包含 150 多种常用流式细胞术试剂的试剂库，这些试剂由机构人员进行预先滴定和等分，供多个 EIB 研究人员使用。试剂库通过批量购买并最大限度地减少表征各个批次试剂所涉及的劳动力和精力来最大限度地降低成本。该设施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目前无法提供的功能。在此期间，该设施竞标并获得资金购买 3 台新的最先进的流式细胞仪。其中两台将取代现有的、过时的仪器，另外一台仪器将增加该设施的容量。