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: 8350096
• 负责人: SUSAN SHARROW
• 金额: $ 172.24万
• 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/8350096
• 关键词: Access to Information; Adhesives; Aliquot; Animals; Antigen-Presenting Cells; Architecture; Area; Arts; B-Cell Activation; Basic Science; Boundary Elements; Cancer Biology; Cancer Research Project; Cell Cycle; Cell Death; Cell Lineage; Cell Separation; Cell Survival; Cell membrane; Cell physiology; Cell surface; Cells; Cellular biology; Child Development; Child health care; Chromatin Structure; Color; Computer software; Consultations; Core Facility; Cost Savings; Cultured Cells; Cytokine Inducible SH2-Containing Protein; Cytokine Signaling; DNA strand break; Data; Data Analyses; Defect; Detection; Development; Electronics; Event; Experimental Designs; Flow Cytometry; Generations; Genetic; Goals; Histocompatibility Antigens; Human; Human Resources; Immune; Immune response; Immunobiology; Immunology; In Vitro; Individual; Inflammation; Inflammatory; Institutes; Integrins; Interleukin 7 Receptor; Investigation; Lasers; Leukocytes; Love; Lymphocyte; MHC Class I Genes; MHC Class II Genes; Manufacturer Name; Measures; Mediating; Membrane; Membrane Microdomains; Metabolic; Metabolism; MicroRNAs; Modification; Molecular; NCI Center for Cancer Research; National Cancer Institute; Pathogenesis; Phosphorylation; Phosphotransferases; Post-Transcriptional Regulation; Problem Solving; Proteins; Reagent; Regulation; Regulation of Exocytosis; Regulatory T-Lymphocyte; Reporting; Research Personnel; Research Project Grants; Research Support; Role; Running; Signal Transduction; Signaling Molecule; Source; Stem cells; Sterility; Stimulus; Structure; T cell differentiation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; TAF7 gene; Techniques; Technology; Tissues; Toll-like receptors; Transcription Initiation; Vaccines; acquired immunity; angiogenesis; anticancer research; base; cost; cytokine; high throughput screening; in vivo; instrument; instrumentation; laser cell sorter; lymphocyte proliferation; member; receptor; receptor expression; release of sequestered calcium ion into cytoplasm; repaired; response; single cell analysis; thymocyte; trafficking

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), Kathleen Kelly (Cell and Cancer Biology Branch, CCR) and Paul Love (National Institute of Child Health and Development). 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 4 user/operator flow cytometers with analysis only capabilities including a state-of-the-art 5-laser flow cytometer analyzer. 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.

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。这些研究涉及对从不同物种和/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进行多参数定量单细胞分析和电子细胞分离。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。核心支持以下 EIB/NCI/CCR 项目： PI：Alfred Singer BC 011106；发育过程中 T 细胞功能的规范 BC 011111；发育中的胸腺细胞和 T 细胞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 细胞分化和库选择 BC 011116； MHC 独立 T 细胞 BC 011117；细胞因子信号转导的 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 细胞库分析 BC 009281；受体介导的 T 细胞和 B 细胞激活 BC 009405；淋巴细胞增殖和复制能力的调节 PI：Andre Nussenzweig BC 010283； DNA 修复 BC 010959； DNA 损伤检测与信号传导之间的关系 BC 010961；DNA 链断裂修复激活剂的高通量筛选 PI：Stephen Shaw 009257 细胞免疫反应机制 010272：促进获取人类蛋白质及其磷酸化信息 010993：淋巴细胞皮质的分子结构 010994：淋巴细胞 近膜嗜碱性激酶 010995：淋巴细胞中的 ERM 磷酸化 010996：ERM 调节的分子机制 PI；黛娜·辛格 BC 009285； MHC I 类基因对外源刺激的反应 SC 010375； TAF7：转录起始中的检查点调节器 BC 010927；染色质结构在调节 MHC I 类表达中的作用 BC 10928；边界元件调节体内 MHC I 类表达 BC 009279； MHC I 类基因表达的调节 PI：David Segal BC 010879； Toll 样受体在获得性免疫产生中的作用 BC 009254； Toll 样受体的结构和功能 PI：Paul Roche BC 009404；抗原呈递细胞中 MHC II 类运输的调节 BC 011033； MHC II 类与质膜微区关联的机制 BC 011035；免疫细胞胞吐作用的调节 PI：Triantafyllos Chavakis BC 010663； JAM-C 在免疫细胞粘附相互作用中的作用 BC 010664；炎症和血管生成之间的串扰 BC 010790；白细胞整合素在炎症细胞募集中的调节 PI：Hyun Park Z1A BC 011214； IL-7 受体表达的转录后调节 Z1A BC 011215；细胞因子信号传导抑制子 (SOCS) 分子的免疫调节作用 在本报告期内，核心还为以下非 EIB PI 提供了有限的研究支持：Thomas Waldmann（CCR 代谢分会）、Jay Berzofsky（CCR 疫苗分会）、Kathleen Kelly（CCR 细胞和癌症生物学分会）和 Paul Love（国家儿童健康与发展研究所）。 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 4 台用户/操作员流式细胞仪，仅具有分析功能，包括一台最先进的 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（包括钙流分析）、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。