Flow Cytometry Laboratory (Scientific Core)

共享资源实验室(SRL)是2003年美国国立卫生研究院路线图的基石。SRL通常被称为核心实验室，是提供来自熟练技术科学家和先进仪器的更广泛科学专门知识的最具成本效益的方法，比个别实验室可行或在经济上可行的范围更广。流式细胞仪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)有利于招聘和保留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)研发支持，6)卫星设施概述，以及7)至少是基于仪器7年生命周期的未来技术扩展和运营的5年计划。 NIA流式细胞仪单位(LMG)作为共享资源实验室(SRL)，为整个NIA IRP提供分析流式细胞仪分析和细胞分类服务。在最近一次审查中，核心被判定为例外。巴尔的摩IRP的每个实验室的研究人员都在使用这个核心。来自核心的99.5%的核心用途、设施和出版物是为了支持NIA内部的科学家。多年来，随着分选能力的提高以及IRP实验室的新研究举措，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，实验室的使用有所增加。需要全天使用多台仪器(2-4台)或更多仪器才能处理超过3-5亿个细胞的用户大量使用分选。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拣需求(50-100个分拣和4亿个待处理的细胞)，使分拣能力得到充分利用，每周4天，其他实验室开放1天。由于需要大量分拣，核心有第4名工作人员负责延长分拣时间，象征性地从下午6点到凌晨4点流动核心是长期纵向研究(格式塔)实验室间检测在免疫学、功能性和表观遗传学水平上改变血细胞亚群(T、B、单核细胞)的重要组成部分。该项目由可持续发展司(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的实验室间项目，专门研究这些动物自然寿命期间老鼠免疫细胞的甲基化和其他表观遗传学变化。 该核心为多参数(多色)分析和分类提供尖端、最先进的流式细胞术。我们的核心是独一无二的，每个主要制造商的仪器。我们拥有唯一的7台激光MoFlo XDP高速分选机和9台iCyt反射式HAPS(高度自动化并行分拣)仪器之一(现有3台HAPS仪器中的1台)。它被安置在贝克BL-2发动机罩中，用于分选腺病毒和慢病毒感染的动物和人类细胞。每个HAP的配置都不同，以便为研究人员的实验设计提供最大的灵活性。我们的MoFlo配备了相干的Genesis MX 460 nm，是半径50英里内唯一能够执行染色体分析和分类的仪器。它还配备了Propel NanoView前向散射探测器，能够检测和分选最小到100 nm的亚微米颗粒，即外显体。BRC设施的实验室空间被设计为在专用的BL-3房间中容纳Aria SORP(4个激光器)，以便能够对活的非人类灵长类细胞进行分选。2015年，我们增加了一台Propel(BioRad)半自动S3分拣机，独一无二地配置了488和640 nm激励。 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，以最佳方式将其先进技术应用于他们的研究，从而提高IRP内的研究范围和质量。这包括拥有一位高素质的主任(拥有36年的流式细胞仪核心经验)，在实验设计、实施和数据解释方面积极咨询，提出新的程序，建议或执行将他们的研究带入新方向的关键实验，以及推动该领域的技术发展。用户可以访问自1999年以来一直在使用的核心网页。 NIA流式细胞术单位的组织和管理方式与校外学术机构和国立卫生研究院的流式细胞术中心的政策和人员配备水平一致。对这些地点(9个国家卫生研究院，133个学术机构，总共142个)的调查(2011年3月更新)表明，91.4%的核心只由核心人员进行分类。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次预定用户分类的能力的级联影响，特别是在核心用户大量预订的情况下)。2)MoFlo和iCyt都不是交钥匙的黑匣子、菜单驱动的仪器(Aria II也不是故障排除专业知识)。 与NIA IRP计划的相关性 除了调查人员对核心的利用程度外，从NIA调查人员自1999年核心建立以来使用流式细胞术/分类作为其研究的一个组成部分的出版物中，可以明显地衡量核心对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.