CCR VB FACS CORE

CCR VB FACS 核心

• 批准号: 8763734
• 负责人: Katherine McKinnon
• 金额: $ 27.28万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763734
• 关键词: Apoptosis; Blood specimen; Bromodeoxyuridine; CCR; Cancer Biology; Cell Cycle; Cell Proliferation; Cells; Collaborations; Color; Computer Systems; Computer software; Containment; Core Facility; Cultured Cells; Event; Experimental Designs; Flow Cytometry; Fluorescent Dyes; Funding; Gene Expression; Goals; HIV; Human; Human T-lymphotropic virus 1; Human T-lymphotropic virus 2; Immunologic Monitoring; In Vitro; Individual; Infectious Agent; Label; Laboratories; Lasers; Life; Monitor; Monoclonal Antibodies; Mus; NCI Center for Cancer Research; National Cancer Institute; Process; Reagent; Receptor Biology and Gene Expression; Research Personnel; Research Project Grants; Retroviridae; Running; SIV; Sampling; Sorting - Cell Movement; Source; Speed; Staining method; Stains; Techniques; Technology; Tissue Sample; Training; Vaccines; Work; analog; biological research; cancer genetics; cellular oncology; cytokine; digital; instrument; instrumentation; meetings; nonhuman primate; polyclonal antibody; rat Ran 2 protein; release of sequestered calcium ion into cytoplasm; research study; single cell analysis

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.

项目概述疫苗分部FACS(流式细胞术)核心设施的创建是为了满足NCI贝塞斯达校区对流式细胞术核心设施的未得到满足的需求，该核心设施具有必要的BL2/BL3容器，用于安全地分选感染了逆转录病毒的活细胞，如HIV/SIV，以及血液和组织样本中已知或怀疑的其他感染源。该设施目前有一台多色细胞分选机，用于标准和传染性分选。此外，该设施还拥有五台多色流式细胞仪的分析能力，这些仪器是由CCR的三名研究人员捐赠给该设施的，并通过IATAP资助。实验室空间的翻修工作于2008年5月完成，捐赠的流式细胞仪于2008年6月迁至该设施。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。这些设备是在09财年初购买的，并于2009财年第二季度安装。该设施目前拥有两台研究人员运行的2台激光4色模拟流式细胞仪，一台研究人员运行的2激光6色数字流式细胞仪，一台研究人员运行的3激光12色数字高端流式细胞仪，以及一台研究人员运行的4激光18色高端数字流式细胞仪。3激光数字高端流式细胞仪进行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，两台激光模拟流式细胞仪都已升级为新的计算机系统。该设施有一台3激光12色数字细胞分选机，配备了用作感染性细胞分选机的容器。截至2009财年第三季度，该分拣机已完全投入使用，并已用于传染性分选。设施经理培训了4名调查员，让他们为各自的项目操作细胞分选机。该设施目前支持来自疫苗分部、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。除了NCI的调查人员外，设施经理还在与FDA的一名调查人员合作。目前在该设施中进行的实验涉及对来自人类、非人类灵长类动物和小鼠原始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。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，以及用于对各种细胞成分进行染色的荧光染料。该设施目前支持的应用包括多色表型分析、细胞周期分析、细胞凋亡分析、BrdU和CFSE增殖分析、细胞内细胞因子分析、罕见事件分析和钙离子通量分析。目前得到支持的项目包括但不限于HIV、SIV、HTLV-1和HTLV-2疫苗在非人类灵长类动物上的免疫监测，以及监测基因表达对细胞增殖和凋亡的结果。