CCR-Frederick Flow Cytometry Core

CCR-Frederick 流式细胞术核心

• 批准号: 7970050
• 负责人: Kathleen Noer
• 金额: $ 120.76万
• 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/7970050
• 关键词: Accounting; Antibodies; Arts; Basic Science; Breast; Cells; Collaborations; Color; Complex; Core Facility; Data; Educational process of instructing; Environment; Epitopes; Family; Flow Cytometry; Future; Genes; Harvest; Hour; Individual; Inflammation; Inflammatory Response; Kidney; Laboratories; Laboratory Scientists; Lasers; Lung; Malignant Neoplasms; Modeling; Mus; NCI Center for Cancer Research; National Cancer Institute; Phycobiliproteins; Phycoerythrin; Population; Principal Investigator; Productivity; Prostate; Proteins; Protocols documentation; Reporter; Research; Research Personnel; Resources; Running; Sampling; Services; Sorting - Cell Movement; Staining method; Stains; Technology; Training; Tumor-Infiltrating Lymphocytes; Update; Work; anticancer research; design; detector; digital; disease characteristic; experience; instrument; instrumentation; meetings; melanoma; member; new technology; novel; programs; research study; tool; tumor

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.

快速变化的流式细胞术领域要求CCR-Frederick Flow Cytotics Core更换旧仪器，并升级我们拥有的新仪器，以满足CCR调查人员的要求。我们在09财年购买了3台新乐器。我们的新LSRII检测16种颜色，具有3个激光和数字数据能力，不久将通过绿色激光增强这一能力，以实现对新的红色荧光报告蛋白质的分析。FACSAria SORP还将配备一种新的绿色激光，这样表达这些新荧光蛋白的细胞就可以被分选出来进行进一步的实验。这些实验将允许使用新的荧光报告蛋白来跟踪几个基因及其相互作用。绿色激光更有效地激发藻红蛋白(一种来自捕光藻胆蛋白家族的红色蛋白质)，它将作为一种非常明亮的荧光色素用于检测模糊表达的表位，如KIR蛋白。绿色激光是应几个CCR实验室的要求购买的，以服务于这些实验室计划在不久的将来进行的项目。两台FACSAria分拣机都将进行流体力学升级，将这些仪器转换为新型号的FACSAria II。旧的BD FACSort将被新的FACSCalibur取代，旧的LSRI将被新的2激光器LSRII Fortessa取代。所有这些新技术都将在今年年底前安装完毕。购买这些新仪器需要更多的空间和新的配置，以适应流式细胞仪核心设施的需要。新的空间经过设计、翻新和工作协调，使仪器的每一步停机时间都减少到只有几个小时。流式细胞术核心工作人员参与了癌症与炎症计划(CIP)范围内合作的设计和分析，以表征来自5个小鼠肿瘤模型的肿瘤浸润性淋巴细胞的炎症反应，使用共同的方案来分解肿瘤和共同的抗体小组，以使结果标准化。这些肿瘤的起源和疾病特征非常不同，因为它们来自小鼠的肺癌、乳腺癌、前列腺癌、肾癌和黑色素瘤。这些数据提供了关于每种癌症的不同微环境的信息，重点是宿主的炎症反应。今年前9个月，流式细胞仪核心工作人员培训了20多名研究人员，让他们自己进行样本操作。工作人员向新手讲授标准的流式细胞仪技术。更有经验的研究人员需要进行复杂的实验来回答他们的研究问题，他们接受了该设施较新的数字仪器的培训，这些仪器能够对更多的抗体组合进行分类。由于这些仪器的复杂性，流式细胞仪的核心工作人员操作所有的细胞分选机。在过去的一年里，我们分类了500多个样品。我们还为那些较少使用这项技术的调查人员(或那些没有接受过运行分析仪培训的人)和那些更喜欢让Core工作人员进行分析的调查人员分析了9000多个样本。通过培训个别研究人员分析自己的样本，Flow Cytotics Core的生产率大大提高。这些乐器每天都在使用，通常是在晚上和周末。在过去的一年里，从37名首席研究人员的实验室为102名科学家个人生成了数据(或对样本进行了分类)。虽然CCR-Frederick流式细胞术核心是CIP的一部分，但该项目的研究人员约占该设施使用的40%。