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: 9154284
• 负责人: SUSAN SHARROW
• 金额: $ 138.33万
• 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/9154284
• 关键词: 12 year old; Acquired Immunodeficiency Syndrome; Aliquot; Animals; Antigen-Presenting Cells; Antigens; Area; Arts; B-Cell Activation; Basic Science; CISH gene; Cancer Research Project; Cancer Vaccines; 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 Receptors; Cytokine Signaling; DNA Damage; DNA Repair; Data; Data Analyses; Defect; Detection; Development; Developmental Biology; Electronics; Event; Experimental Autoimmune Encephalomyelitis; Experimental Designs; Family member; Flow Cytometry; Gene Expression Regulation; Generations; Genetic; Genetic Transcription; Goals; Histocompatibility Antigens; Human Resources; Immune; Immunobiology; Immunology; In Vitro; Individual; Interleukin 7 Receptor; Interleukin-15; Interleukin-2; Investigation; Kidney Diseases; Laboratories; Lasers; Love; Lymphocyte; MHC Class I Genes; MHC Class II Genes; Manufacturer Name; Measures; Mediating; Membrane Microdomains; Metabolic; Metabolism; MicroRNAs; Modification; NCI Center for Cancer Research; National Cancer Institute; National Institute of Child Health and Human Development; National Institute of Diabetes and Digestive and Kidney Diseases; Pathogenesis; Pathology; Phosphotransferases; Post-Transcriptional Regulation; Problem Solving; 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; T cell differentiation; T-Cell Activation; T-Cell Development; T-Cell Receptor; T-Lymphocyte; TAF7 gene; Techniques; Technology; Tissues; Transcription Initiation; Vaccine Design; Vaccines; anticancer research; base; cancer therapy; cost; cytokine; genome integrity; in vivo; instrument; instrumentation; laser cell sorter; lymphocyte proliferation; member; receptor; receptor expression; release of sequestered calcium ion into cytoplasm; 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 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: Damian Kovalovsky ZIA BC 011429 Zbtb family members in lymphocyte differentiation and function. PI: Andre Nussenzweig Z1A BC 010283 DNA repair, Z1A BC 010959 Relationship between DNA damage detection and signaling. PI: Jay Berzofsky Jay ZIA SC 004020 Antigen-specific T-cell Activation, Application to Vaccines for Cancer and AIDS. PI: Thomas Waldmann ZIA SC 004002 IL-2/IL-15 Cytokine Receptor: Implications for Cancer Therapy and Vaccine Design. PI: David Levens Z1A BC 011465. During this reporting period, the facility provided limited research support to the following non-EIB CCR PI: Thomas Waldmann (Metabolism Branch, CCR), Jay Berzofsky (Vaccine Branch, CCR), and David Levens (Laboratory of Pathology, CCR) and continues to provide support to prior EIB member, Andre Nussenzweig now Chief, Laboratory of Genome Integrity CCR. In addition the facility has provided limited support to non-NCI PI Jeffrey Kopp, Kidney Diseases Branch, NIDDK and Paul Love, Section on Cellular and Developmental Biology, NICHD. 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. In November 2013, the Facility replaced a 12 year old 1-laser user instrument with a new, state of the art, 3 laser user instrument. 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 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细胞库生成的研究; g）移植抗原表达的分析; h）T细胞谱系发育中涉及的机制的研究; i）细胞死亡的机制; j）干细胞分析和k）免疫基因调节的机制。以下EIB/NCI/CCR项目得到了核心的支持：PI：Alfred Singer; ZIA BC 009273 T细胞分化和库选择，ZIA BC 011106发育期间T细胞功能的规范，ZIA BC 011111发育中的胸腺细胞和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，Z1 A BC 011113 T细胞存活，Z1 A BC 011114 microRNA在T细胞发育中的作用，Z1 A BC 011116 MHC非依赖性T细胞，Z1 A BC 011117 T细胞受体调节细胞因子信号传导。主要研究者：理查德·霍兹：Z1 A BC 009265; T细胞库分析，Z1 A BC 009281受体介导的T和B细胞活化，Z1 A BC 009405;淋巴细胞增殖和复制能力的调节。主要研究者：Dinah Singer Z1A BC 009285 Responses of MHC Class I Genes to Exogenous 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.主要研究者：Paul Roche Z1 A BC 009404抗原呈递细胞中MHC II类分子运输的调节，Z1 A BC 011033 MHC II类分子与质膜微区结合的机制，Z1 A BC 011035免疫细胞胞吐作用的调节。主要研究者：Hyun Park Z1 A BC 011214白细胞介素-7受体表达的转录后调节，Z1 A BC 011215细胞因子信号转导抑制因子（SOCS）分子的免疫调节作用。主要研究者：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通过微小RNA调节TH 17细胞中的T-bet表达。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 Z1 A BC 010283 DNA修复，Z1 A BC 010959 DNA损伤检测和信号传导之间的关系。PI：Jay Berzofsky Jay ZIA SC 004020抗原特异性T细胞活化，应用于癌症和艾滋病疫苗。PI：托马斯瓦尔德曼ZIA SC 004002 IL-2/IL-15细胞因子受体：对癌症治疗和疫苗设计的意义。PI：大卫莱文斯Z1 A BC 011465。在本报告期内，该机构为以下非EIB CCR 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·伯佐夫斯基（疫苗分支，CCR）和大卫莱文斯（病理学实验室，CCR），并继续为前EIB成员Andre Nussenzweig（现任基因组完整性实验室CCR主任）提供支持。此外，该机构还向非NCI PI Jeffrey Kopp（NIDDK肾脏疾病分支）和Paul Love（NICHD细胞和发育生物学部门）提供了有限的支持。该机构运行并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）。2013年11月，该设施将使用了12年的单激光用户仪器更换为新的最先进的三激光用户仪器。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、试剂选择和数据分析与解释。该设施支持各种流式细胞术应用，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。作为一项节约成本的措施，该机构还维护了一个包含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源提供的软件目前无法提供的功能。