CCR VB FACS CORE

项目概述疫苗分支FACS（流式细胞术）核心设施的创建是为了满足NCI Bethesda校区对具有BL 2/BL 3防护的流式细胞术核心设施的未满足需求，该防护是安全分选受逆转录病毒（如HIV/SIV）以及血液和组织样本中已知或疑似的其他感染因子感染的活细胞所必需的。该设施有一台目前用于标准和感染性分选的细胞分选机。此外，该设施还拥有五台流式细胞仪的分析能力，这些仪器是CCR的三名研究人员捐赠给该设施的，并通过IATAP提供资金。2008年5月完成了实验室空间的翻修，捐赠的流式细胞仪于2008年6月搬迁到该设施。该机构购买了一台4激光高端分析仪和一台3激光高速细胞分选仪。这些是在2009财年初购买的，并在2009财年第二季度安装。 该机构目前拥有两台双稳态运行2激光4色模拟流式细胞仪、一台双稳态运行2激光6色数字流式细胞仪、一台双稳态运行3激光12色数字高端流式细胞仪和一台双稳态运行4激光18色高端数字流式细胞仪。3台激光数字高端流式细胞仪已进行了广泛的硬件和软件升级，以增加额外的荧光和样本处理能力，2台激光模拟流式细胞仪已升级为新的计算机系统。该设施有一个3激光12色数字细胞分选仪，配备有作为感染性细胞分选仪使用的容器。该分拣机已全面投入使用，并已用于2009财年第3季度的感染性分拣。机构经理培训了4名研究者，以操作其各自项目的细胞分选机。该设施目前支持来自疫苗分支、受体生物学和基因表达实验室、细胞肿瘤学实验室以及癌症生物学和遗传学实验室的70多名NCI/CCR研究人员。除NCI调查员外，设施经理还与FDA的一名调查员合作。 目前在该设施中进行的实验涉及来自人类、非人类灵长类动物和小鼠原始来源的细胞的多参数单细胞分析。此外，还对体外培养的细胞进行了分析。用于这些实验的试剂包括具有荧光标记的单克隆和多克隆抗体以及用于染色多种细胞组分的荧光染料。该设施目前支持的应用包括多色表型分析、细胞周期分析、凋亡分析、BrdU和CFSE增殖分析、细胞内细胞因子分析、罕见事件分析和钙通量分析。目前支持的项目包括但不限于对非人灵长类动物的HIV、SIV、HTLV-1和HTLV-2疫苗研究的免疫监测，以及监测基因表达对细胞增殖和凋亡的结果。

Project Summary The Vaccine Branch FACS (Flow Cytometry) Core Facility has been created to meet an unmet need on the Bethesda Campus of NCI for a Flow Cytometry Core Facility with BL2/BL3 containment necessary to safely sort live cells infected with retroviruses, such as HIV/SIV, and other infectious agents known or suspected in blood and tissue samples. The facility has a multicolor cell sorter currently in use for standard and infectious sorting. In addition, this facility has analysis capabilities from five multicolor flow cytometry instruments that were donated to the facility from three Investigators in the CCR, and through IATAP funding. Laboratory space renovations were completed in May 2008, and the donated flow cytometers were relocated to the facility in June 2008. The facility purchased a 4 laser high end analyzer and a 3 laser high speed cell sorter. These were purchased at the beginning of FY09 and installed in Q2 FY09. The facility currently maintains two investigator-run 2 laser 4 color analog flow cytometers, one investigator-run 2 laser 6 color digital flow cytometer, one investigator-run 3 laser 12 color digital high end flow cytometer, and one investigator-run 4 laser 18 color high end digital flow cytometer. The 3 laser digital high end flow cytometer has undergone extensive hardware and software upgrades to add additional fluorescent and sample processing abilities, and both of the 2 laser analog flow cytometers have been upgraded to a new computer system. The facility has a 3 laser 12 color digital cell sorter equipped with containment for use as an infectious cell sorter. The sorter is fully operational and has been used for infectious sorting as of Q3 FY09. The Facility Manager has trained 4 investigators to operate the cell sorter for their individual projects. The facility currently supports more than 70 NCI/CCR Investigators from the Vaccine Branch, the Laboratory of Receptor Biology and Gene Expression, the Laboratory of Cellular Oncology, and the Laboratory of Cancer Biology and Genetics. In addition to NCI investigators, the Facility Manager is working on a collaboration with an investigator from the FDA. Experiments currently conducted in the facility involve multi-parameter single cell analysis of cells from human, non-human primate and mouse primary sources. Additionally analysis is also conducted on in vitro cultured cells. Reagents for these experiments include monoclonal and polyclonal antibodies with fluorescent labels and fluorescent dyes used for staining a variety of cellular components. Current applications being supported by the facility include multi-color phenotypic analysis, cell cycle analysis, apoptosis analysis, proliferation analysis with BrdU and CFSE, intracellular cytokine analysis, rare event analysis and calcium flux analysis. Currently supported projects include, but are not limited to, immune monitoring for HIV, SIV, HTLV-1 and HTLV-2 vaccine studies on non-human primates, and monitoring the results of gene expression on cell proliferation and apoptosis.