Flow Cytometry Laboratory (Scientific Core)

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，是最具成本效益的方法，可以从熟练的技术科学家和先进的仪器中提供更广泛的科学专业知识，而不是单个实验室的可行性或经济可行性。流式细胞术SRL的最佳实践包括：1）稳定和一致的财政支持，2）高素质的科学主任（负责人），3）有利于招聘和留住高技能技术人员的政策，4）用户培训和工作人员继续教育，5）仪器质量控制计划，6）支持研究和开发，6）卫星设施概述，以及7）至少为未来的技术扩展和操作制定一个5年计划，该计划基于仪器的7年生命周期。 NIA流式细胞仪单元（LMG）作为共享资源实验室（SRL）为整个NIA IRP提供流式细胞仪分析和细胞分选服务。在上次审查中，核心被认为是例外。该核心是由来自巴尔的摩IRP的每个基于实验室的研究人员使用的。99.5%的核心使用，设施和来自核心的出版物都是为了支持NIA的内部科学家。随着IRP实验室的新研究计划（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），分选能力沿着增加，多年来实验室的使用量有所增加。分选被需要多个仪器（2-4个）全天或更长时间以便处理超过3亿至5亿个细胞的用户大量使用。一些研究人员的实验方案需要特定的仪器。除了目前的核心用户外，另外两个实验室预计在不久的将来会有大量的分选需求（50-100种和超过4亿个细胞的处理），这使得分选能力每周有4天得到充分利用，其中1天开放给其他实验室。由于需要大量分拣，核心有第四名工作人员负责延长分拣时间，从下午6点到凌晨4点。流动核心是长期、纵向研究（完形）实验室间的一个组成部分，在免疫、功能和表观遗传水平上检查血细胞亚群（T、B、单核细胞）的变化。这一项目由国家执行局国际研究项目司发起，利用了很大一部分核心资源。该核心还参与了另一个长期的实验室间项目，专门研究大鼠免疫细胞在这些动物的自然寿命中的甲基化和其他表观遗传变化。 该核心为多参数（多色）分析和分选提供了最先进的流式细胞术。 我们的核心是独特的仪器从每个主要制造商。我们拥有唯一的7激光MoFlo XDP高速分选机和9台iCyt反射式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）。 它被安置在Baker BL-2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都有不同的配置，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 460 nm，是50英里半径内唯一能够进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100 nm的亚微米颗粒，即外来体。BRC设施的实验室空间设计用于在专用BL-3室中容纳Aria SORP（4台激光器），以便能够分选活的非人类灵长类动物细胞。2015年，我们增加了Propel（BioRad）半自动S3分选机，独特配置了488和640 nm激发。 NIA流式细胞仪共享资源实验室的使命是通过为所有研究人员提供必要的专业知识，以最佳方式利用其先进技术进行研究，从而提高IRP内研究的范围和质量。这包括拥有一位高素质的主任（拥有36年的流式细胞术核心经验），对实验设计，实施和数据解释进行积极的咨询，制定新的程序，建议或执行关键实验，将他们的研究推向新的方向，并推进该领域的技术发展。用户可以访问自1999年以来一直使用的核心网页。 NIA流式细胞术单位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备水平一致。对这些站点（9个NIH站点，133个学术站点，总共142个站点）的调查（2011年3月更新）表明，在91.4%的核心中，仅由核心人员进行排序。这样做的原因是双重的：1）这是向IRP提供可靠服务的最有效的方式（即，最小化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排序的能力的级联效应，特别是当核心被大量预订时）。2)MoFlo和iCyt都不是一个可供使用的黑盒、菜单驱动的仪器（Aria II也不是专门用于故障排除的仪器）。 与NIA IRP计划的相关性 除了研究者的核心利用水平外，自1999年核心开始以来，NIA研究者使用流式细胞术/分选作为其研究的一个组成部分所产生的出版物也证明了核心对IRP生产力的有效性和价值。

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.