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)干细胞分析和k)免疫基因调控机制。以下EIB/NCI/CCR项目由核心支持：PI: Alfred Singer；ZIA BC 009273 T细胞分化和库选择，ZIA BC 011106 T细胞发育过程中的功能规范，Z1A BC 011111细胞因子信号在发育中的作用，Z1A BC 011111调节性T细胞的发育和功能，Z1A BC 011113 T细胞存活，Z1A BC 011114 microrna在T细胞发育中的作用，Z1A BC 011116 mhc非依赖性T细胞，Z1A BC 011117 T细胞受体对细胞因子信号的调节。PI：理查德·霍兹：Z1A BC 009265；T细胞库分析，Z1A BC 009281受体介导的T和B细胞活化，Z1A BC 009405淋巴细胞增殖和复制能力的调控。Z1A BC 009285 MHC I类基因对外源刺激的响应，Z1A SC 010375 TAF7：一个转录起始的检查点调节器，Z1A BC 009279 MHC I类基因的表达调控，ZIA BC 011381 Brd4是一个调节转录的非典型激酶，ZIA BC 011425在Treg细胞中的表达。抗原呈递细胞中MHCⅱ类转运的调控，MHCⅱ类与质膜微结构域的关联机制，免疫细胞胞外分泌的调控。白介素-7受体表达的转录后调控，细胞因子信号传导抑制因子（SOCS）分子的免疫调节作用。T-bet在实验性自身免疫性脑脊髓炎发病机制中的作用，微rna调控TH17细胞中T-bet的表达。Zbtb家族成员在淋巴细胞分化及功能中的作用PI: Andre Nussenzweig Z1A BC 010283 DNA修复，Z1A BC 010959 DNA损伤检测与信号传导的关系。抗原特异性t细胞活化在癌症和艾滋病疫苗中的应用。IL-2/IL-15细胞因子受体在肿瘤治疗和疫苗设计中的应用[j]。PI: David Levens Z1A BC 011465。在本报告所述期间，该设施为以下非EIB CCR PI提供了有限的研究支持：Thomas Waldmann（代谢科，CCR）， Jay Berzofsky（疫苗科，CCR）和David Levens（病理实验室，CCR），并继续为之前的EIB成员Andre Nussenzweig（现任基因组完整性CCR实验室主任）提供支持。此外，该设施还向非nci PI Jeffrey Kopp （NIDDK肾病科）和Paul Love （NICHD细胞和发育生物学科）提供了有限的支持。该设施操作和维护两台具有细胞分选能力的操作员操作多激光流式细胞仪，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12年的1激光用户仪器。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领域向调查人员提供咨询。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; 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.