CCR-Frederick Flow Cytometry Core

为了满足CCR研究人员的要求，流式细胞术领域的快速变化要求流式核心取代旧仪器并升级我们现有的新仪器。在2010财年早期，FACSAria SORP和LSRII SORP都添加了绿色激光器，以促进红色荧光蛋白的分选和分析。研究人员一直在忙于开发表达红色荧光蛋白的转基因小鼠模型，因此刚刚开始使用绿色激光检测转基因小鼠中附着其感兴趣基因的红色荧光蛋白。其他人已经发现，它可以很好地检测低表达蛋白，如Kir与藻红蛋白结合抗体。越来越多的研究人员增加了实验的复杂性，并常规地使用6-9抗体进行实验，以确定不同肿瘤模型中肿瘤浸润淋巴细胞的不同群体。利用9种抗体对各种罕见事件肿瘤浸润淋巴细胞进行分类，确定群体，通过定量RT-PCR检测参与肿瘤炎症反应的RNA表达。荧光探测器数量的增加大大提高了每次实验中关于炎症在肿瘤进展中的作用的信息的质量和复杂性。至少应该有一名研究者使用12种颜色来定义他们感兴趣的肿瘤浸润淋巴细胞群，以确定肿瘤中细胞因子的表达和调节炎症反应。flow核心人员在今年前三个季度培训了16名研究人员来运行他们自己的样本，向流式细胞术的新手教授旧技术，而需要复杂实验来回答他们的研究问题的更有经验的研究人员则接受了使用具有更多抗体组合能力的新数字仪器的培训。这些仪器每天都由训练有素的调查人员使用，经常使用到深夜和周末。这使得流量核心人员有更多的时间在今年前三个季度对大约450个样品进行分类，并分析大约7200个样品。如果没有调查者获取数据并分析他们自己的样本，政府将不得不雇佣一个或两个以上有经验的全职人员来完成同样数量的工作。在过去的一年中，至少有6篇论文发表了使用在流动岩心中获得的数据。在过去的一年里，已经为来自37个主要研究人员的实验室的102名科学家生成了数据（或分类了样本）。虽然CCR-Frederick流式细胞仪核心是CIP的一部分，但来自该项目的研究人员约占该设备使用的47%。未来的目标包括通过扩大经过培训的研究人员的数量，以运行和分析弗雷德里克所有CCR的自己的样品，从而扩大使用核心仪器和专业知识产生的科学的使用，生产力和质量，从而跟上流动实验室的尖端技术。这可以通过在LSRII Fortessa上增加一台405激光器来实现，用小型2激光6色仪器取代两台BD facscan。

The fast changing field of flow cytometry has required the flow core to replace old instrumentation and up-grade the newer instruments that we have in order to meet the requirements of our CCR investigators. In early FY10 a green laser was added to both the FACSAria SORP and the LSRII SORP to facilitate the sorting and analysis of red fluorescent proteins. Investigators have been busy developing the transgenic mouse models with red fluorescent protiens expressed and have therefore just begun to use the green laser to detect red fluorescent proteins with their gene of interest attached in transgenic mice. Others have found that it detects low expression proteins such as Kir very well with a Phycoerythrin conjugated antibody. Many more of the investigators have increased the complexity of their experiments and are routinely running experiments with 6-9 antibodies to define distinct populations of tumor infiltrating lymphocytes in different tumor models. Various rare event tumor infiltrating lymphocytes have been sorted using 9 antibodies to define the populations in order to detect RNA expression involved in the inflammatory response to the tumor measured by quantitative RT-PCR. The larger number of fluorescence detectors has greatly increased the quality and complexity of information generated about the role of inflammation in tumor progression with each experiment. There should be at least one investigator working on using 12 colors to define their tumor infiltrating lymphocyte populations of interest to determine the cytokines expressed and regulating the inflammation response in tumors. The flow core staff has trained sixteen investigators in the first three quarters of the year to run their own samples teaching the older technology to the more novice in flow cytometry while the more experienced investigators with the need for complex experiments to answer their research questions have been trained on the newer digital instruments with the capability of more antibody combinations. The instruments are used daily by the trained investigators often into the night and on the weekends. This leaves more time for the flow core staff to sort approximately 450 samples and analyze about 7200 samples in the first three quarters of this year. Without the investigators acquiring the data and analyzing their own samples, the government would have to hire one or more probably two full time experienced individuals to perform the same volume of work. At least 6 papers have been published in the past year using data obtained in the flow core. 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 47% of the use of this facility. The future goals include keeping up with cutting-edge technology in the flow lab by expanding the pool of investigators trained to run and analyze their own samples to all of CCR in Frederick thereby expanding the use, productivity and quality of the science generated using the core's instrumentation and expertise. This could be achieved by adding a 405 laser to the LSRII Fortessa, replacing the two BD FACScans with small 2 laser 6-color instruments.