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)用户培训和工作人员继续教育，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。从2011年平衡计分卡的审查中，该核心被判定为例外。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，如分选小鼠和人类肌肉干细胞，实验室的使用量有所增加。2012财年核心使用率为2011年容量的102%，2013财年迁移到BRC之前为101%。用户需要使用多台仪器(2-3台)进行全天或更长时间的分选，才能处理3亿个细胞。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。 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。流式细胞仪的使用寿命为57年，为了规划未来，IRP在2008年购买了一台iCyt Reflect HAPS(高度自动化并行分选)仪器，该仪器安装在Baker BL-2引擎盖中，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。2013年，随着转向BRC，过时的MoFlo高速细胞分选机(2001年购买，部件已不再广泛使用)升级为数字版本，除了从模拟仪器保留的640 nm激光器和90-5水冷Ar和氪离子激光器外，还包括405 nm、532 nm和561 nm激光。我们目前在使用该仪器时遇到了问题。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。服务合同成本约占年度运营预算的80%，随着MoFlo超出保修期，该成本将在2014年增加。 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32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，建议或执行关键实验，将他们的研究带入新的方向，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都不是交钥匙黑盒、菜单驱动的仪器(Aria II也不是故障排除能力方面的仪器)。 随着仪器容量的增加，分拣的使用也得到了扩展。在2011财年和2012财年，内核的运行速度为容量的102%。 与NIA IRP计划的相关性 除了调查人员对核心的利用程度外，自1999年核心建立以来，从NIA调查人员使用流式细胞术/分类作为其研究的一个组成部分产生的157份出版物中，可以明显地衡量核心对IRP生产力的有效性和价值。 这些期刊的部分清单包括J免疫学(10)、BMC免疫学(2)、J Exp Med(1)、细胞免疫学(2)、免疫学(1)、自然免疫学(1)、血液(2)、癌症Res(1)、PNAS USA(3)、内分泌学(1)、PLOSone(3)、DNA修复(3)、Mol Cell Biol(3)、EMBO J(1)和自然(1)，以及其他期刊(43)。使用核心服务的研究论文有98.5%来自NIA IRP调查人员。

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. 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 from the 2011 BSC 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 murine and human muscle stem cells. FY12 core usage was at 102% of capacity 2011 capacity and until the move to the BRC was 101% for FY2013. Sorting is heavily scheduled by users requiring multiple instruments (2-3) for the full day or greater in order to process >300 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. The core provides cutting edge, state-of-the-art flow cytometry for multi-parameter (polychromatic) analysis and sorting. Flow cytometry instrumentation has a 5 7 year lifespan and to plan for the future, the IRP has made major instrument purchases in 2008 with the acquisition of an iCyt Reflection HAPS (Highly Automated Parallel Sorting) instrument that 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. In 2013 with the move to the BRC, the obsolete (purchased in 2001 with parts no longer widely available) MoFlo high speed cell sorter was upgraded to an digital version incorporating 405nm, 532nm, and 561nm lasers in addition to an 640nm laser and the 90-5 water-cooled argon and krypton-ion lasers that were retained from the analog instrument. We are currently experiencing problematic issues with this instrument. 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. Service contract costs account for approximately 80% of the annual operating budget and will increase in 2014 as the MoFlo comes out of its warranty period. 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 32yrs 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 the ability to troubleshoot). Sort usage has expanded in parallel with instrument capacity. For FY11 and FY 12, the core ran at 102% of capacity. 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 157 publications generated by NIA investigators using flow cytometry/sorting as an integral part of their research since the cores inception in 1999. A partial list of these includes J Immunology (10), BMC Immunology (2), J Exp Med (1), Cellular Immunology (2), Immunity (1), Nature Immunology (1), Blood (2), Cancer Res (1), PNAS USA (3), Endocrinology (1), PLOSone (3), DNA Repair (3), Mol Cell Biol (3), EMBO J (1), and Nature (1), other journals (43). 98.5% of research papers using core services are from NIA IRP investigators.