Application of Flow Cytometry to Cell Biology

实验免疫学分部(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。这些研究包括对新鲜从不同物种和/或组织制备的细胞以及体外培养细胞的光谱进行多参数定量单细胞分析和基于与细胞相关的参数的电子细胞分离。基础研究支持提供给EIB成员和NCI癌症研究中心内的其他研究人员。目前得到支持的项目包括但不限于以下研究领域：a)通过细胞表面分子进行的细胞内信号传递的体内和体外分析；b)动物和/或基因修饰细胞的细胞过程和/或缺陷的分析；c)免疫发病机制和后果的研究；d)各种分子的细胞表面配位表达的分析；e)T细胞库的研究；g)移植抗原的表达分析；h)T细胞谱系发育的机制的研究；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细胞分化和谱系选择；Zia BC 011106发育中T细胞功能规范；Z1a BC 011111胸腺细胞和T细胞发育中的细胞因子信号转导；Zia BC 011112调节性T细胞的发展和功能；Z1a BC 011113 T细胞生存；Z1ABC 011114微RNA在T细胞发育中的作用；Z1ABC 011116 MHC非依赖T细胞；Z1ABC 011117 T细胞受体对细胞因子信号的调节。Pi：Richard Hodes：Z1ABC 009265；T细胞谱系分析，Z1ABC 009281受体介导的T和B细胞激活，Z1ABC 009405；淋巴细胞增殖和复制能力的调节。PI：Dinah Singer Z1ABC 009285 MHC I类基因对外界刺激的反应，Z1ASC 010375 TAF7：转录启动的检查点调节因子，Z1ABC 009279 MHC I类基因表达的调控，ZIA BC 011381 Brd4是一种非典型的调节转录的激酶，ZIA BC 011425在Treg细胞中表达I类。Pi：Paul Roche Z1a BC 009404对MHC II类抗原呈递细胞运输的调节，Z1a BC 011033 MHC II类与质膜微域相关的机制，Z1a BC 011035调节免疫细胞的胞吐作用。PI：Hyun Park Z1ABC 011214转录后对白细胞介素7受体表达的调节，Z1ABC 011215免疫调节细胞因子信号转导(SOCS)分子的作用。Pi：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microRNA调节T-bet在TH17细胞中的表达。PI：Damian Kovalovsky Zia BC 011429 ZBTB家族成员的淋巴细胞分化和功能。Pi：Andre Nussenzweig Z1ABC 010283 DNA修复，Z1ABC 010959 DNA损伤检测与信号传递的关系。PI：Jay Berzofsky Jay Zia SC 004020抗原特异性T细胞激活，应用于癌症和艾滋病疫苗。PI：Thomas Red Zia BC 010835结直肠癌转录组：基因组不稳定性和非整倍体的影响。PI：Thomas Waldmann Zia SC 004002 IL-2/IL-15细胞因子受体：对癌症治疗和疫苗设计的影响。在本报告所述期间，该机构向以下非EIB CCR PI提供了有限的研究支持：Thomas Waldmann(新陈代谢科)、Jay Berzofsky(疫苗科，CCR)、Thomas Red(遗传学科)，并继续向以前的EIB成员、现任CCR基因组完整性实验室主任的Andre Nussenzweig提供支持。此外，该设施还为NIDDK肾脏病科的非NCI Pi Jeffrey Kopp和NICHD细胞和发育生物学科的Paul Love提供了有限的支持。该设施运营和维护两台操作员运行的具有细胞分选功能的多激光流式细胞仪，包括一台最先进的6激光细胞分选机和5台仅具有分析功能的用户/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。2013年11月，该设施用新的、最先进的3激光用户仪器取代了已有12年历史的1激光用户仪器。设施工作人员为研究人员提供以下方面的咨询：实验设计、解决问题、试剂选择以及数据分析和解释。该设施支持广泛的流式细胞仪应用，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，该设施还保留了一个试剂库，其中包括150多种常用的流式细胞仪试剂，这些试剂由设施人员预先命名和等量，供多名EIB调查人员使用。试剂库通过批量购买并最大限度地减少鉴定单个批次试剂所涉及的人力和精力来最大限度地降低成本。该机构正在开发用于流式细胞仪分析的基于Windows的PC软件，该软件将提供仪器制造商或第三方软件来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; 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 Ried ZIA BC 010835 Colorectal Cancer Transcriptome: Effects of Genomic Instability and Aneuploidy. PI: Thomas Waldmann ZIA SC 004002 IL-2/IL-15 Cytokine Receptor: Implications for Cancer Therapy and Vaccine Design. 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), Thomas Ried, (Genetics Branch, 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.