CCR-Frederick Flow Cytometry Core

CCR-Frederick 流式细胞术核心

• 批准号: 8158374
• 负责人: Kathleen Noer
• 金额: $ 88.68万
• 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/8158374
• 关键词: Flow Cytometry

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.

快速变化的流式细胞术领域要求流式核心更换旧仪器并升级我们拥有的新仪器，以满足CCR研究人员的要求。在2010财年早期，FACSAria SORP和LSRII SORP都增加了绿色激光，以促进红色荧光蛋白的分选和分析。研究者们一直在忙碌忙着开发表达红色荧光蛋白的转基因小鼠模型，因此，利用绿色激光检测转基因小鼠中附着有目的基因的红色荧光蛋白才刚刚开始。其他人已经发现，它用藻红蛋白缀合的抗体很好地检测低表达蛋白质，如Kir。更多的研究者增加了他们实验的复杂性，并且常规地使用6-9种抗体进行实验，以在不同的肿瘤模型中定义不同的肿瘤浸润淋巴细胞群体。使用9种抗体对各种罕见事件肿瘤浸润淋巴细胞进行分选，以确定群体，从而检测通过定量RT-PCR测量的肿瘤炎症反应中涉及的RNA表达。大量的荧光检测器极大地提高了每次实验产生的关于炎症在肿瘤进展中的作用的信息的质量和复杂性。至少应有一名研究者使用12种颜色定义其感兴趣的肿瘤浸润淋巴细胞群，以确定肿瘤中表达的细胞因子和调节炎症反应。在今年的前三个季度，流式细胞术核心人员培训了16名研究人员运行自己的样本，向流式细胞术的新手教授较旧的技术，而需要复杂实验来回答其研究问题的较有经验的研究人员则接受了具有更多抗体组合能力的较新数字仪器的培训。受过训练的调查员每天使用这些仪器，经常使用到深夜和周末。这为流量核心人员留出了更多时间，在今年前三季度对约450个样本进行分类，并对约7200个样本进行分析。如果没有调查人员获取数据并分析自己的样本，政府将不得不雇用一个或多个（可能是两个）全职经验丰富的人来完成同样数量的工作。在过去的一年中，至少有6篇论文使用了在流动核心中获得的数据。 在过去一年中，为37名主要调查员的实验室的102名科学家生成了数据（或对样本进行了分类）。尽管CCR-Frederick流式细胞术核心是CIP的一部分，但该项目的研究者占该设施使用量的约47%。未来的目标包括通过将经过培训的调查人员队伍扩大到弗雷德里克的所有CCR，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。