CCR-Frederick Flow Cytometry Core

快速变化的流式细胞术领域需要CCR-Frederick Flow Cytometry Core用于替换旧仪器并升级我们现有的新仪器 以满足CCR研究者的要求。我们在2009财年购买了3台新仪器。我们的新 LSRII可检测16种颜色，具有3个激光器和数字数据功能，不久将在 用绿色激光增强，以能够分析新的红色荧光报告物 proteins. FACSAria SORP还将配备一种新的绿色激光器， 表达这些新的荧光蛋白的细胞可以被分选用于进一步的实验。这些 实验将允许跟踪几个基因及其相互作用 使用新的荧光报告蛋白。绿色激光激发藻红蛋白（一种红色 来自捕光藻胆蛋白家族的蛋白质）更有效地，这将是 可用作非常明亮的荧光染料，以检测微弱表达的表位，例如Kir 蛋白绿色激光器是应几个CCR实验室的要求获得的， 这些实验室计划在不久的将来进行的项目。两台FACSAria分拣机 将进行射流升级，将这些仪器转换为新型号FACSAria 二.旧的BD FACSort将被新的FACSCalibur取代，旧的LSRI将被 更换为新的双激光LSRII Fortessa。所有这些新技术都将由 今年年底这些新仪器的购置需要更多的空间， 配置以满足流式细胞术核心设施的需求。新的空间是 设计，翻新和协调工作，以便在每一步仪器停机时间 最小化到几个小时流式细胞术核心工作人员参与设计 和分析癌症和炎症计划（CIP）范围内的合作，以表征 用免疫组织化学方法检测5种小鼠肿瘤模型肿瘤浸润淋巴细胞的炎症反应 一个共同的协议来分解肿瘤和一个共同的抗体面板， 将被标准化。这些肿瘤有非常不同的起源和疾病特征 因为它们来源于小鼠的肺癌、乳腺癌、前列腺癌、肾癌和黑色素瘤。这 数据已经提供了关于这些癌症中的每一种的不同微环境的信息， 关注宿主的炎症反应今年首九个月， 流式细胞术核心工作人员培训了20多名研究者运行自己的样本。工作人员 成员们向新用户传授标准流式细胞术技术。更有经验 研究人员需要复杂的实验来回答他们的研究问题， 他们接受了该设施更新的数字仪器的培训，这些仪器能够对更多的 抗体组合。流式细胞术核心工作人员操作所有细胞分选仪， 这些工具的复杂性。在过去的一年里，我们对500多个样本进行了分类。 我们还分析了9000多个样本，为那些调查人员谁使用这种技术少 通常（或未接受过运行分析仪培训的人员）和 我更愿意让核心人员进行分析。流式细胞术的生产率 通过培训个别调查人员分析自己的数据， 样品这些仪器每天都在使用，经常在晚上和周末使用。过去 2009年，为来自世界各地的102名科学家生成了数据（或对样本进行了分类）。 37名主要研究人员的实验室。尽管CCR-Frederick流式细胞仪核心 是CIP的一部分，来自该计划的研究人员约占使用量的40% 这个设施。

The rapidly changing field of flow cytometry has required the CCR-Frederick Flow Cytometry Core to replace old instrumentation and upgrade the newer instruments that we have to meet the requirements of CCR investigators. We purchased 3 new instruments in FY09. Our new LSRII detects 16 colors, with 3 lasers and digital data capabilities that will soon be enhanced with a green laser to enable the analysis of the new red fluorescent reporter proteins. The FACSAria SORP will also be equipped with a new green laser so that cells expressing these new fluorescent proteins can be sorted for further experiments. These experiments will allow several genes and their interactions with each other to be tracked using the new fluorescent reporter proteins. The green laser excites phycoerythrin (a red protein from the light-harvesting phycobiliprotein family) more efficiently, which will be useful as a very bright fluorchrome to detect dimly expressed epitopes such as the Kir protein. The green lasers were acquired at the request of several CCR laboratories to serve projects that these labs are planning to conduct in the near future. Both FACSAria sorters will have fluidics upgrades that will convert these instruments into the new model FACSAria II. The old BD FACSort will be replaced with a new FACSCalibur and the old LSRI will be replaced with a new 2-laser LSRII Fortessa. All of this new technology will be installed by the end of this year. The acquisition of these new instruments required more space with a new configuration to fit the needs of the Flow Cytometry Core facility. The new space was designed, renovated, and the work coordinated so that at each step instrument downtime was minimized to just a few hours. Flow Cytometry Core staff members were involved in the design and analysis of a Cancer and Inflammation Program (CIP)-wide collaboration to characterize the inflammatory response of tumor-infiltrating lymphocytes from 5 mouse tumor models using a common protocol to disaggregate the tumors and a common antibody panel so that the results would be standardized. These tumors have very different origins and disease characteristics because they are derived from mouse lung, breast, prostate, renal, and melanoma cancers. This data has provided information on the diverse micro-environments of each of these cancers, focusing on the inflammatory response of the host. During the first 9 months of this year, the Flow Cytometry Core staff trained more than 20 investigators to run their own samples. Staff members taught the standard flow cytometry technology to novice users. More experienced investigators, who require complex experiments to answer their research questions, were trained on the facility's newer digital instruments, which are capable of sorting more antibody combinations. Flow Cytometry Core staff members operate all of the cell sorters due to the complexity of these instruments. During the last year, we sorted more than 500 samples. We also analyzed more than 9000 samples for those investigators who use this technology less often (or those who are not trained to run the analyzers) and for those investigators who prefer to have the Core staff conduct the analysis. The productivity of the Flow Cytometry Core has been greatly increased by training individual investigators to analyze their own samples. The instruments are used daily, often into the night and on the weekends. In the past year, data has been generated (or samples have been sorted) for 102 individual scientists from the laboratories of 37 principal investigators. Although the CCR-Frederick Flow Cytometry Core is part of the CIP, investigators from this program account for approximately 40% of the use of this facility.