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: 8554067
• 负责人: SUSAN SHARROW
• 金额: $ 133.77万
• 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/8554067
• 关键词: Access to Information; Aliquot; Animals; Antigens; Architecture; Area; Arts; Autoimmune Process; B-Lymphocytes; Basic Science; Boundary Elements; Cancer Biology; Cancer Research Project; Cell Cycle; Cell Death; Cell Lineage; Cell Separation; 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 Damage; DNA Repair; DNA strand break; Data; Data Analyses; Defect; Detection; Development; Electronics; Event; Experimental Designs; Family member; Flow Cytometry; Gene Expression Regulation; Generations; Genetic; Genetic Transcription; Genome; Goals; Histocompatibility Antigens; Human; Human Resources; Immune; Immunobiology; Immunology; In Vitro; Individual; Institutes; Interleukin 7 Receptor; Investigation; Laboratories; Lasers; Love; Lymphocyte; MHC Class I Genes; MHC Class II Genes; Manufacturer Name; Measures; Mediating; Membrane; Metabolic; Metabolism; MicroRNAs; Modification; Molecular; NCI Center for Cancer Research; National Cancer Institute; Pathogenesis; Phosphorylation; Post-Transcriptional Regulation; Problem Solving; Proteins; Reagent; Regulation; Regulation of Exocytosis; Reporting; Research Personnel; Research Project Grants; Research Support; Role; Running; Signal Transduction; Source; Stem cells; Sterility; Structure; T cell differentiation; T-Cell Receptor; T-Lymphocyte; TAF7 gene; Techniques; Technology; Tissues; Toll-like receptors; Vaccines; anticancer research; base; cost; cytokine; high throughput screening; in vivo; instrument; instrumentation; laser cell sorter; lymphocyte proliferation; member; receptor; 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; 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.

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）T细胞谱系生成的研究; g）移植抗原表达的分析; 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;发育中胸腺细胞和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细胞分化和谱系选择BC 011116; MHC非依赖性T细胞BC 011117; T细胞受体对细胞因子信号传导的调节BC 011112;调节性T细胞的发育和功能PI：Richard HodesBC 009265; T细胞谱系分析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：细胞免疫应答的机制010272：促进人类蛋白质及其磷酸化信息的获取010993：淋巴细胞皮质的分子结构010994：淋巴细胞膜近端嗜碱性激酶010995：淋巴细胞中的ERM磷酸化010996：ERM调节的分子机制PI; Dinah SingerBC 009285; MHC I类基因对外源刺激的反应SC 010375; TAF 7：转录起始中的检查点调节因子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：大卫SegalBC 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 RocheBC 009404; MHC II类分子在抗原呈递细胞中运输的调节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 ParkZ 1A BC 011214;白细胞介素-7受体表达的转录后调节Z1 A BC 011215;细胞因子信号传导抑制因子（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ZIA BC 011432通过microRNAs PI：Damian Kovalovsky ZIA BC 011429; Zbtb家族成员在淋巴细胞分化和功能中的作用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在本报告期间，核心还为以下非EIB PI提供了有限的研究支持：托马斯瓦尔德曼（代谢分支，CCR），杰伊Berzofsky（疫苗分支，CCR），凯瑟琳凯利（细胞和癌症生物学分支，CCR）和保罗爱（国家儿童健康和发展研究所）。核心还继续向安德烈·努森茨韦格提供有限的支持（基因组完整性实验室，该设施操作和维护两台操作员运行的具有细胞分选能力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，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。 该设备支持多种流式细胞仪应用，包括：（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、包括钙流分析的代谢分析、无菌细胞分选和细胞内细胞因子分析。该设施还作为成本节约措施，维护一个包含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。该设施正在开发基于WINDOWS的流式细胞术分析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。