Flow Cytometry Laboratory (Scientific Core)

流式细胞术实验室（科学核心）

基本信息

批准号：
9550595
负责人：
Robert Wersto
金额：
$ 82.9万
依托单位：
NATIONAL INSTITUTE ON AGING
依托单位国家：
美国
项目类别：
财政年份：
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/9550595
关键词：
Algorithms Animals Antigens Apoptosis Apoptotic Baltimore Biological Assay Blood Cells Bone Marrow Bone Marrow Stem Cell Books Bromodeoxyuridine Cardiac Myocytes Cell Cycle Cell Separation Cell surface Cells Chromosomes Color Complex Computers and Advanced Instrumentation Consultations Continuing Education Data Analyses Detection Development Discrimination Effectiveness Epigenetic Process Event Experimental Designs Extramural Activities Financial Support Flow Cytometry Flow Cytometry Shared Resource Frequencies Future Generations Head Hour Human Human Resources Immune Immunofluorescence Immunologic Immunologics Individual Institution Laboratories Lasers Life Cycle Stages Longevity Longitudinal Studies MC 540 Manufacturer Name Measurement Methylation Mission Mitosis Modeling Modification Mus Muscle satellite cell Neurons Peripheral Blood Lymphocyte Pharmaceutical Preparations Physiologic pulse Policies Population Population Analysis Procedures Process Productivity Propidium Diiodide Proteins Protocols documentation Publications Quality Control RNA Radial Rattus Recruitment Activity Reporter Reporter Genes Research Research Personnel Research Support Resource Sharing Resources Sampling Schedule Science Scientist Services Side Site Sorting - Cell Movement Speed Spleen Staining method Stains Stem cells Subfamily lentivirinae Surveys Technical Expertise Technology Training United States National Institutes of Health Update Visit base c-myc Genes cost effective daughter cell design detector embryonic stem cell exosome experience experimental study flexibility follow-up human embryonic stem cell innovation instrument instrumentation member monocyte nerve stem cell nonhuman primate novel operation particle programs research and development submicron thymocyte web page

项目摘要

Shared Resources Laboratories (SRL) are fundamental cornerstones of the 2003 NIH Roadmap. Often referred to as core laboratories, SRLs are the most cost-effective approach to provide a broader range of scientific expertise from skilled technology scientists and advanced instrumentation than would be feasible or economically-viable for individual laboratories. Best practices for a Flow Cytometry SRL include: 1) stable and consistent financial support, 2) a highly qualified scientific director (head), 3) policies conducive to recruitment and retention of highly skilled technical staff, 4) user training and staff continuing education, 5) a quality control program for the instruments, 6) support for research and development, 6) overview of satellite facilities, and 7) at a minimum, a 5-year plan for future technology expansion and operation based on a 7yr life cycle for instrumentation. The NIA Flow Cytometry Unit (LMG) functions as a Shared Resource Laboratory (SRL) to provide analytical flow cytometric analysis and cell sorting services to the entire NIA IRP. The core was judged to be exceptional in its last review. The core is used by investigators from every bench-science based laboratory in the Baltimore IRP. 99.5% of the core usage, facilities, and publications coming from the core are in support of NIA intramural scientists. Laboratory usage has increased over the years as sorting capacity has increased along with new research initiatives by IRP labs, such as sorting human and mouse immune cells, murine muscle stem cells and bone marrow stem cells. Sorting is heavily utilized by users that require multiple instruments (2-4) for the full day or greater in order to process in excess of 300 - 500 million cells. Some investigators experimental protocols require specific instruments. Besides the current core users, two additional laboratories have projected substantial sorting needs (50-100 sorts and >400 million cells for processing) in the near future, obligating full use of the sorting capacity for 4 days per week with 1 day open for other laboratories. Because of need for high volume sorting, the core has a 4th staff member who is responsible for extended hours sorting, nominal from the hours of 6PM to 4AM. The flow core is an integral part of long-term, longitudinal study (Gestalt) inter-laboratory examining changes blood cell subpopulations (T, B, monocyte) at the immunological, functional and epigenetic levels. This project, initiated by the SD, NIA IRP, utilizes a substantial portion of the core resources. The core is also involved in another long-term interlaboratory project specifically examining methylation and other epigenetic changes in rat immune cells over the natural lifespan of these animals. The core provides cutting edge, state-of-the-art flow cytometry for multi-parameter (polychromatic) analysis and sorting. Our core is unique with instrumentation from each major manufacturer. We have the only 7-laser MoFlo XDP high speed sorter and one of 9 iCyt Reflection HAPS (Highly Automated Parallel Sorting) instruments (1 of 2 of the 3 HAPS instruments in existence). It is housed in a Baker BL-2 hood for sorting adeno- and lentivirus-infected animal and human cells. Each HAPS is configured differently to provide the maximum flexibility for investigators experimental design. Our MoFlo is equipped with a Coherent Genesis MX 460nm, and is the only instrument in a 50 mile radius able to perform chromosome analysis and sorting. It is also equipped with a Propel NanoView forward scatter detector, enabling detection and sorting of submicron particles in the down to 100nm, i.e exosomes. The laboratory space for the BRC facility was designed to house the Aria SORP (4 lasers) in a dedicated BL-3 room to be able to sort live non-human primate cells. In 2015, we added a Propel (BioRad) semi-automated S3 sorter, uniquely configured with both 488 and 640nm excitation. The mission of the NIA flow cytometry shared resource lab is to enhance the scope and quality of the research within the IRP by providing all investigators with the expertise necessary to employ its advanced technologies optimally for their research. This includes having a highly qualified director (with 36yrs flow cytometry core experience), active consultation on experimental design, implementation, and data interpretation, originating new procedures, suggesting or performing the critical experiments that take their research in new directions, and in advancing technological developments in the field. Users can visit the core web page, in use since 1999. The NIA Flow Cytometry Unit is organized and managed in a manner consistent with the policies and staffing levels of flow cytometry cores at extramural academic institutions and at NIH. A survey (updated March 2011) of these sites (9 NIH, 133 academic, total 142) indicates that in 91.4% of the cores, sorting is performed only by core personnel. The reason for this is twofold: 1) it is the most efficient way to provide reliable services to the IRP (i.e. minimizing the possibility of instrument abuse by users and thus removing the cascading effect that this has on the ability to complete the next scheduled users sort, especially when the core is heavily booked). 2) neither the MoFlo nor iCyt are turnkey black box, menu-driven instruments (nor is the Aria II in the case of troubleshooting expertise). Relevance to the NIA IRP Program In addition to the level of core utilization by investigators, measurement of the effectiveness and value of the core to IRP productivity is evident from the publications generated by NIA investigators using flow cytometry/sorting as an integral part of their research since the cores inception in 1999.

共享资源实验室 (SRL) 是 2003 年 NIH 路线图的基本基石。 SRL 通常被称为核心实验室，它是最具成本效益的方法，可以提供比单个实验室可行或经济上可行的更广泛的科学专业知识，这些专业知识来自熟练的技术科学家和先进的仪器。流式细胞仪 SRL 的最佳实践包括：1) 稳定且一致的财务支持，2) 高素质的科学总监（负责人），3) 有利于招聘和留住高技能技术人员的政策，4) 用户培训和员工继续教育，5）仪器的质量控制计划，6）对研究和开发的支持，6）卫星设施概述，以及 7）至少基于 7 年生命周期的未来技术扩展和运营的 5 年计划为了仪器仪表。 NIA 流式细胞术单元 (LMG) 作为共享资源实验室 (SRL)，为整个 NIA IRP 提供分析流式细胞术分析和细胞分选服务。该核心在上次审查中被评为优秀。巴尔的摩 IRP 的每个基础科学实验室的研究人员都在使用该核心。来自核心的 99.5% 的核心使用、设施和出版物都是为了支持 NIA 校内科学家。多年来，随着分选能力的提高以及 IRP 实验室的新研究举措（例如分选人类和小鼠免疫细胞、小鼠肌肉干细胞和骨髓干细胞），实验室的使用量有所增加。用户大量使用分选，需要全天或更长时间使用多台仪器 (2-4) 才能处理超过 300 - 5 亿个细胞。一些研究人员的实验方案需要特定的仪器。除了当前的核心用户外，另外两个实验室预计在不久的将来会有大量分选需求（50-100 种分选和超过 4 亿个细胞用于处理），要求每周 4 天充分利用分选能力，其中 1 天开放给其他用户实验室。由于需要进行大量分拣，核心有一名 4 名工作人员负责延长分拣时间，名义上从下午 6 点到凌晨 4 点。流程核心是长期纵向研究 (Gestalt) 实验室间检查免疫学、功能和表观遗传水平上血细胞亚群（T、B、单核细胞）变化的一个组成部分。该项目由 SD、NIA IRP 发起，利用了大部分核心资源。该核心还参与了另一个长期实验室间项目，专门检查大鼠免疫细胞在这些动物自然寿命期间的甲基化和其他表观遗传变化。该核心提供最先进的流式细胞术，用于多参数（多色）分析和分类。我们的核心是独一无二的，拥有来自各大制造商的仪器。我们拥有唯一的 7 激光 MoFlo XDP 高速分选机和 9 台 iCyt 反射 HAPS（高度自动化并行分选）仪器之一（现有 3 台 HAPS 仪器中的 2 台之一）。它安装在 Baker BL-2 防护罩中，用于分选腺病毒和慢病毒感染的动物和人类细胞。每个 HAPS 的配置都不同，为研究人员的实验设计提供最大的灵活性。我们的 MoFlo 配备了 Coherent Genesis MX 460nm，是 50 英里半径内唯一能够执行染色体分析和分选的仪器。它还配备了 Propel NanoView 前向散射检测器，能够检测和分选低至 100nm 的亚微米颗粒，即外泌体。 BRC 设施的实验室空间旨在将 Aria SORP（4 个激光器）放置在专用的 BL-3 房间中，以便能够对活的非人类灵长类动物细胞进行分类。 2015 年，我们添加了 Propel (BioRad) 半自动 S3 分选机，独特配置了 488 和 640 nm 激发。 NIA 流式细胞术共享资源实验室的使命是通过为所有研究人员提供最佳利用其先进技术进行研究所需的专业知识，来提高 IRP 内研究的范围和质量。这包括拥有一位高素质的主管（拥有 36 年流式细胞术核心经验），积极咨询实验设计、实施和数据解释，制定新程序，建议或执行关键实验，将他们的研究带向新方向，并推动技术进步该领域的发展。用户可以访问自 1999 年以来使用的核心网页。 NIA 流式细胞术部门的组织和管理方式与校外学术机构和 NIH 流式细胞术核心的政策和人员配置水平一致。对这些网站（9 个 NIH、133 个学术网站、总共 142 个）的调查（2011 年 3 月更新）表明，在 91.4% 的核心中，分类仅由核心人员执行。这样做的原因有两个：1) 这是向 IRP 提供可靠服务的最有效方式（即最大限度地减少用户滥用仪器的可能性，从而消除这对完成下一个预定用户的能力所产生的级联效应）排序，特别是当核心被大量预订时）。 2) MoFlo 和 iCyt 都不是交钥匙黑匣子、菜单驱动仪器（就故障排除专业知识而言，Aria II 也不是）。与 NIA IRP 计划的相关性除了研究人员的核心利用率水平之外，自 1999 年核心启动以来，NIA 研究人员使用流式细胞术/分选作为其研究的一个组成部分，所发表的出版物中也可以明显看出对核心对 IRP 生产力的有效性和价值的衡量。