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和其他癌症研究中心（CCR）的50多名研究人员的多个研究项目。这些研究涉及多参数定量单细胞分析和基于不同物种和/或组织新鲜制备的细胞相关参数的电子细胞分离，以及体外培养细胞的光谱。基础研究支持提供给EIB成员和NCI癌症研究中心的其他研究人员。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的体内和体外分析；B)动物和/或基因修饰细胞的细胞过程和/或缺陷分析；C)免疫发病机制和后果的研究；D)各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)研究涉及T细胞谱系发育的机制；1)细胞死亡机制；j)干细胞分析。以下EIB/NCI/CCR项目由核心支持：PI: Alfred Singer BC 011106；T细胞发育过程中的功能规范细胞因子信号在胸腺细胞和T细胞发育中的作用T细胞存活BC 011114；microrna在T细胞发育中的作用T细胞分化和细胞库选择；mhc非依赖性T细胞BC 011117；T细胞受体对细胞因子信号通路的调控调节性T细胞的发育与功能[j]；T细胞库BC 009281的分析受体介导的T细胞和B细胞活化BC 009405淋巴细胞增殖和复制能力的调节[j]； Andre Nussenzweig BC 010283；Dna修复BC 010959；dna损伤检测与信号传导BC 010961的关系高通量筛选DNA链断裂修复激活因子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：淋巴细胞皮层分子结构010994：淋巴细胞膜近端嗜碱性激酶010995：淋巴细胞ERM磷酸化010996:ERM调控的分子机制PI；黛娜辛格BC 009285；MHC I类基因对外源刺激SC 010375的反应TAF7：转录起始BC 010927的检查点调节器染色质结构在MHCⅰ类表达调控中的作用边界元件调控MHC I类在体内的表达MHC I类基因的表达调控[j]；toll样受体在获得性免疫BC 009254产生中的作用toll样受体的结构与功能；MHCⅱ类在抗原呈递细胞BC 011033中的转运调控MHCⅱ类与质膜微域BC 011035关联机制的研究免疫细胞胞吐调节机制的研究JAM-C在免疫细胞BC 010664粘附相互作用中的作用炎症与血管生成BC 010790的串扰白细胞整合素在炎症细胞募集中的调控作用白细胞介素-7受体Z1A BC 011215的转录后调控在本报告期间，核心还为以下非eib PI提供了有限的研究支持：Thomas Waldmann（代谢分支，CCR）， Jay Berzofsky（疫苗分支，CCR）， Kathleen Kelly（细胞和癌症生物学分支，CCR）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，具有细胞分选能力，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，仅具有分析能力，包括一台最先进的5激光流式细胞仪分析仪。设施工作人员在实验设计、问题解决、试剂选择和数据分析和解释等方面为调查人员提供咨询。该设备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。作为一项节省成本的措施，该设施还维护了一个包含150多种常用流式细胞仪试剂的试剂库，这些试剂由设施人员预滴定和引用，供多个EIB研究人员使用。试剂库通过批量购买和最大限度地减少人工和努力表征单个批次试剂的成本。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.