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。该设施操作和维护2台具有细胞分选功能的操作人员操作的3台激光流式细胞仪和3台仅具有分析功能的用户操作流式细胞仪。该设施最近获得了最先进的5激光分析流式细胞仪，将用于扩展和扩大可用的应用。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 facility operates and maintains 2 operator run 3-laser flow cytometers with cell sorting capabilities and 3 user-operator flow cytometers with analysis only capabilities. The facility has recently acquired a state-of-the-art 5-laser, analysis only, flow cytometer that will be used to extend and expand available applications. 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.