CCR VB FACS CORE

疫苗分部FACS（流式细胞术）核心设施的创建是为了满足NCI Bethesda校区对BL2/BL3遏制的流式细胞术核心设施的未满足需求，以安全分选感染逆转录病毒（如HIV/SIV）和其他已知或怀疑存在于血液和组织样本中的感染因子的活细胞。该设施有一个多色细胞分选器，目前用于标准和传染性分选。此外，该设施还具备5台多色流式细胞仪的分析能力，这5台多色流式细胞仪是由CCR的3位研究人员和IATAP基金捐赠给该设施的。实验室空间翻修于2008年5月完成，捐赠的流式细胞仪于2008年6月搬迁到该设施。该设施购买了一台4激光高端分析仪和一台3激光高速细胞分选机。这些是在2009财年初购买的，并在2009财年第二季度安装。该设施目前拥有两台研究人员运行2激光4彩色模拟流式细胞仪，一台研究人员运行2激光6彩色数字流式细胞仪，一台研究人员运行3激光12彩色数字高端流式细胞仪和一台研究人员运行4激光18彩色高端数字流式细胞仪。3型激光数字高端流式细胞仪经过了广泛的硬件和软件升级，以增加额外的荧光和样品处理能力，2型激光模拟流式细胞仪都升级到新的计算机系统。该设施有一台3激光12色数字细胞分选机，配有容器，用作感染性细胞分选机。该分选机已全面投入使用，并已于2009财年第三季度用于感染性分选。该设施目前支持来自疫苗处、受体生物学和基因表达实验室以及细胞肿瘤学实验室的50多名研究人员。由于该设施开业仅4个月，预计调查人员将大幅增加。目前在该设施进行的实验包括对来自人类、非人类灵长类动物和小鼠主要来源的细胞进行多参数单细胞分析。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带有荧光标记的单克隆和多克隆抗体和用于染色各种细胞成分的荧光染料。该设备目前支持的应用包括多色表型分析、细胞周期分析、细胞凋亡分析、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类灵长类动物的HIV、SIV、HTLV-1和HTLV-2疫苗的免疫监测，以及对细胞增殖和凋亡的基因表达结果的监测。

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 currently supports more than 50 Investigators from the Vaccine Branch, the Laboratory of Receptor Biology and Gene Expression, and the Laboratory of Cellular Oncology. Since the facility has only been open for 4 months, the number of investigators is expected to increase significantly. 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.