Flow cytometry support to research in immune, skin, muscle and bone diseases

该设施目前支持大多数NIAMS IRP研究人员的研究。此外，它还积极参与仪器培训和流式细胞仪检测开发，为所有不熟悉该技术的IRP用户提供服务。 该基金目前通过以下仪器支持NIAMS IRP社区： (One)BD Biosciences FACSAria Fusion细胞分选仪（5激光激发，19色检测，自动化细胞沉积单元，生物安全柜）新购买，以取代MoFlo细胞分选仪 (One)BD Biosciences FACSAria IIIu细胞分选仪（5激光激发，19色检测，自动细胞沉积单元） (One)BD Biosciences Influx细胞分选仪（7激光激发，20色检测，自动细胞沉积单元，光谱分析仪） (One)Beckman Coulter MoFlo细胞分选仪（3激光激发，10色检测，自动细胞沉积装置）在2014年6月实验室搬迁前使用了10多年后退役 (One)BD Biosciences FACSCanto SORP分析仪，带HTS（4激光激发，14色检测，高通量进样器） (One)BD Biosciences LSR Fortessa分析仪（7激光激发，20色检测，高通量进样器） (Two)BD Biosciences FACSVerse分析仪（3激光激发，8色检测，通用高通量进样器，体积测量（用于绝对细胞计数）） (One)Compucyte iCys research激光扫描细胞仪（3激光激发，5参数检测） (One)Amnis ImageStreamX Mark II高分辨率显微镜/细胞仪（7激光激发，3成像物镜，分析软件） 除了这些实验室侧重于免疫系统的机制和功能障碍的项目外，该设施还继续向NIAMS实验室提供流式细胞术和分选服务，这些实验室从事流式细胞术不常服务的学科。 最近增加了FACSAria Fusion细胞分选仪，该设备具有层流生物安全柜，现在可以更安全地使用该设备分选患者标本和人源细胞。10号楼内的核心设施从6D搬迁到10 C也提供了将BD InFlux细胞分选机放置在Baker生物安全柜中的机会。这些分类器现在为NIAMS研究人员提供了更好的机会，以进一步推动NIH主任弗朗西斯柯林斯博士推动的“从实验室到临床”倡议。 Influx细胞分选仪和LSR Fortessa分析仪配备了7个激光器和20多个荧光检测器，继续提供最先进的工具，使研究人员能够利用最新的荧光染料和荧光蛋白报告基因。 那些研究涉及不适合流式细胞术的标本的研究人员可以使用该部门的激光扫描细胞仪，Thorlabs（Compucyte）iCys以及Amnis ImageStreamx Mark II。这些基于显微镜的仪器提供固定在载玻片或多孔板（iCys）上的细胞或组织的荧光和形态学数据，或如传统流式细胞术（ImageStream）中的悬浮细胞。沿着典型的免疫表型分析、细胞凋亡测定和共定位研究，iCys还可以随着时间的推移重新分析特定细胞，用于基于动力学的测定。相比之下，ImageStream Mark II的设计允许更高的吞吐量，以获得更稳健的统计数据，这是标准流式细胞仪的一个特征，同时提供基于显微镜的系统的图像分析。 FCS有三名专职人员支持NIAMS IRP调查员的需求： 先生杰弗里·莱自2010年以来一直是该部门的一员。他最近接受了使用Amnis ImageStream的培训，并将继续推广和支持该仪器的使用。 Kevin Tinsley博士于2013年1月初作为承包商加入核心设施。在完成了免疫学领域的博士学位和博士后研究后，他加入了核心，对流式细胞术非常熟悉。他于2013年在BD Biosciences完成了FACSAria培训，现在是FACSAria IIIu的主要操作员。 James Simone先生在履行部门负责人职责的同时，继续参与NIH和中大西洋地区的流式细胞术推广工作。作为流式细胞术兴趣小组的联合主席，他协助组织和推广季度会议，展示该领域的领先研究人员，并促进与流式细胞术相关产品供应商的关系。他长期担任国际细胞计数促进协会（ISAC）和ISAC生物安全委员会的成员，有助于促进与世界各地同行交流新的想法和信息。 迄今为止，已有200多名NIAMS流式细胞仪用户在该设施提供的不同平台上接受了培训。我们继续成功使用2011年推出的多层次培训计划。提供一个更合理的培训结构，一个渐进的小组和个人为基础的培训系统被用来提供研究者和他们的工作人员更好地获得流式细胞术科的设备和服务。所有新用户都参加了对设施设备和政策的一般介绍和指导。然后，根据需要和以往的经验，他们进展到最适合他们的特定研究需要的仪器的个性化或小组教学。经部门领导批准后，用户即可在无人协助的情况下操作仪器。但是，在所有情况下，FCS工作人员都可以进行故障排除和咨询。

The facility currently supports the research of the majority of NIAMS IRP investigators. In addition, it is actively involved in instrument training and flow cytometric assay development for all IRP users unfamiliar with this technique. The Facility currently supports the NIAMS IRP community with the following instrumentation: (One) BD Biosciences FACSAria Fusion cell sorter (5-laser excitation, 19-color detection, automated cell deposition unit, Biosafety Cabinet) new purchase to replace MoFlo cell sorter (One) BD Biosciences FACSAria IIIu cell sorter (5-laser excitation, 19-color detection, automated cell deposition unit) (One) BD Biosciences Influx cell sorter (7-laser excitation, 20-color detection, automated cell deposition unit, spectral analyzer (One) Beckman Coulter MoFlo cell sorter (3-laser excitation, 10-color detection, automated cell deposition unit) retired after 10+ years of service prior to laboratory relocation in June 2014 (One) BD Biosciences FACSCanto SORP analyzer w/HTS (4-laser excitation, 14-color detection, high-throughput sampler) (One) BD Biosciences LSR Fortessa analyzer (7-laser excitation, 20-color detection, high-throughput sampler) (Two) BD Biosciences FACSVerse analyzers (3-laser excitation, 8-color detection, universal high-throughput sampler, volumetric measurement (for absolute cell counts) (One) Compucyte iCys research laser scanning cytometer (3-laser excitation, 5-parameter detection) (One) Amnis ImageStreamX Mark II high resolution microscope/cytometer (7-laser excitation, 3 imaging objectives, analysis software) In addition to the projects from those laboratories focused on mechanisms and dysfunctions of the immune system, the facility has continued to provide flow cytometry and sorting services to NIAMS laboratories engaged in disciplines not often served by flow cytometry. Usage of the facility to sort patient specimens and human-derived cells can now be done with a higher degree of safety with the recent addition of the FACSAria Fusion cell sorter, which features a laminar flow biosafety cabinet. The core facility relocation within Bldg. 10 from 6D to 10C also provided the opportunity to house the BD InFlux cell sorter in a Baker biosafety cabinet, as well. These sorters now provide better opportunities for NIAMS investigators to further the Bench to Bedside initiative promoted by NIH Director, Dr. Francis Collins. Equipped with seven lasers and 20+ fluorescence detectors, the Influx cell sorter and LSR Fortessa analyzers continue to provide state-of-the-art tools, enabling investigators to take advantage of the latest fluorochromes and fluorescent protein reporters. Those investigators whose research involves specimens that are not well suited to flow cytometry can utilize the Section's laser scanning cytometer, the Thorlabs (Compucyte) iCys, as well as the Amnis ImageStreamx Mark II. These microscope-based instruments provide fluorescent and morphological data on cells or tissue fixed to slides or multi-well plates (iCys) or cells in suspension as in traditional flow cytometry (ImageStream). Along with typical immunophenotyping, apoptosis assays and co-localization studies, the iCys can also re-analyze specific cells over time for kinetics-based assays. In contrast, the design of the ImageStream Mark II allows for higher throughput for more robust statistics, a characteristic of standard flow cytometers, while providing image analysis of a microscope-based system. The FCS has three dedicated personnel to support the need of NIAMS IRP investigators: Mr. Jeffrey Lay has been a part of the section since 2010. He has been recently trained to use the Amnis ImageStream and will continue to promote and support usage of the instrument. Kevin Tinsley, PhD, joined the core facility in early January 2013 as a contractor. Having completed his doctoral degree and postdoctoral fellowship in the field of immunology, he joined the Core with a substantial familiarity with flow cytometry. He completed his FACSAria training at BD Biosciences in 2013 and is now the primary operator of the FACSAria IIIu. While fulfilling his duties as Section Leader, Mr. James Simone continues to be involved in the promotion of flow cytometry at the NIH and within the Mid-Atlantic Region. As a co-chair of the Flow Cytometry Interest Group, he assists in organizing and promoting quarterly meetings that showcase leading researchers in the field and promote relationships with vendors of flow cytometry-related products. His longtime membership in the International Society for the Advancement of Cytometry (ISAC) and the ISAC Bio-Safety Committee helps facilitate the exchange of new ideas and information with peers from around the world. More than two hundred NIAMS flow cytometry users have been trained to date on the different platforms available in the facility. We continue to use successfully the multi-level training scheme that was introduced in 2011. Providing a more logical training structure, a progressive group and individual-based training system is used to provide investigators and their staff better access to the Flow Cytometry Sections equipment and services. All new users attend a general introduction and orientation to facility equipment and policies. Then, according to needs and previous experience, they progress to individualized or group instruction on the instruments most appropriate for their particular research needs. After approval by the section leader, users can then operate the instruments unassisted. In all cases, however, the FCS staff is available for troubleshooting and consultation.