Application of Flow Cytometry to Cell Biology

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;和j）干细胞分析。以下EIB/NCI/CCR项目由核心支持：PI：Alfred Singer BC 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;细胞因子信号传导的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：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细胞免疫应答机制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 Singer BC 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剂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; I类MHC基因表达的调节PI：大卫西格尔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：保罗罗氏BC 009404;抗原呈递细胞中II类MHC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：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;炎症细胞募集中白细胞整合素的调节PI：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;细胞因子信号传导抑制因子（SOCS）分子的免疫调节作用在本报告期间，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）。 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。

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.