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

摘要： 这一细胞学核心设备支持大多数NIAMS IRP研究人员的研究。此外，它还积极参与为这项技术的新用户提供的细胞仪培训、数据分析和流式细胞仪分析开发。 该科目前拥有和维护以下仪器，以供国家航空航天监测系统使用： -(1)BD Bioscience FACSAria融合细胞分选机(5激光激发、19色检测、自动细胞沉积装置、生物安全柜) -(1)BD Bioscience FACSAria IIIu细胞分选机(5激光激发、19色检测、自动细胞沉积单元) -(1)BD Bioscience Inflation细胞分选仪(7-激光激发、20色检测、自动细胞沉积仪、光谱分析仪) -(1)BD Bioscience FACSCanto SORP分析仪，带HTS(4激光激发、14色检测、高通量采样器) -(1)BD Bioscience LSR Fortessa SORP分析仪，带HTS(7激光激发、20色检测、高通量采样器) -(2)BD Bioscience FACSVerse分析仪(S)，(3激光激发，8色检测，通用高通量采样器多孔板或管，绝对细胞计数体积测量) -复合式ICYS激光扫描细胞仪(3激光激发，5参数检测)，与NCI共享 -AMNIS ImageStreamX Mark II高分辨率成像细胞仪(7个激光激发、3个成像物镜、IdeasTM分析软件) 在过去的几年里，功能界别核心安置和使用了其他细胞仪(如下所列)，以支持NIAMS的任务。自那以后，这些乐器已经退役，取而代之的是上面列出的更现代的乐器。 -Beckman Coulter MoFlo高速细胞分选器 -Beckman Coulter青色ADP分析仪 -BD Biosciences FACSCalibur -BD Biosciences FACS Vantage DIVA高速细胞分选机 -BD Biosciences LSR I 除了这些实验室专注于免疫系统机制和功能障碍的项目外，该设施还继续为NIAMS实验室提供流式细胞仪和分类服务，这些实验室从事的学科不是很容易被流式细胞仪服务，如骨骼、肌肉和皮肤生物学。 该设施还具有以更高的安全性对患者样本和人类来源细胞进行分类的能力。随着FACSAria Fusion细胞分选机最近的增加，安装在层流生物安全柜中的设施现有的BD Income细胞分选机在2014年6月的核心设施搬迁期间也同样获得了自己的生物安全柜。这些细胞分选器现在为NIAMS的调查人员提供了更安全的机会，以进一步推动由NIH主任Francis Collins博士推动的从板凳到床边的倡议。 每个都配备了7个用于激发的激光器和20个单独的荧光检测器，流入细胞分选器和LSR Fortessa分析仪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程序。 那些研究涉及的标本不太适合流式细胞术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Amennis ImageStreamX Mark II。这些基于显微镜的仪器提供细胞、固定在玻片上的组织、多孔板(ICY)或悬浮细胞的荧光和形态数据，与传统的流式细胞仪(ImageStreamX)相同。除了典型的免疫表型、凋亡分析和共定位研究外，ICYS还可以随着时间的推移重新分析特定细胞，进行基于动力学的分析。相比之下，ImageStreamX Mark II的设计允许更高的吞吐量，以获得更可靠的统计数据，这是标准流式细胞仪的特点，同时提供基于显微镜的系统的图像分析优势。 金融统计框架有三名专职人员支持NIAMS IRP调查人员的需求： --先生。自2010年以来，杰弗里·雷一直是该部门的一部分。他最近接受了使用Amny ImageStreamX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，现在是员工科学家。在完成免疫学领域的博士学位和博士后研究后，他加入了Core，拥有丰富的流式细胞仪经验。他随后于2013年在BD生物科学公司完成了FACSAria操作员培训，现在是FACSAria IIIu的主要操作员。 --先生。科长James Simone负责管理该设施的日常运营，并继续参与NIH和大西洋中部地区的流式细胞术推广工作。作为美国国立卫生研究院流式细胞术兴趣小组的联合主席，他协助组织和推动季度会议，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学促进会(ISAC)和国际细胞学促进会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。 到目前为止，已有250多名NIAMS流式细胞仪用户接受了关于该设施现有的各种细胞仪的培训。所有新用户都参加了设施设备和政策的一般介绍和培训。然后，根据需要和以前的经验，他们就最适合他们特定研究需要的仪器进行个性化或小组指导。在得到科长的批准后，用户可以在没有辅助的情况下操作仪器。然而，在所有情况下，功能界别工作人员都可以提供技术支持和咨询。 为了进一步支持NIAMS的任务，流式细胞科继续研究细胞学中的新技术和应用，以供其调查人员使用。

Summary: This cytometry core facility supports the research of the majority of NIAMS IRP investigators. Moreover, it is actively involved in cytometer training, data analysis and flow cytometric assay development for users new to this technique. The Section currently houses and maintains the following instrumentation for the use of NIAMS: -(1) BD Biosciences FACSAria Fusion cell sorter (5-laser excitation, 19-color detection, automated cell deposition unit, Biosafety Cabinet) -(1) BD Biosciences FACSAria IIIu cell sorter (5-laser excitation, 19-color detection, automated cell deposition unit) -(1) BD Biosciences Influx cell sorter (7-laser excitation, 20-color detection, automated cell deposition unit, spectral analyzer) -(1) BD Biosciences FACSCanto SORP analyzer w/HTS (4-laser excitation, 14-color detection, high-throughput sampler) -(1) BD Biosciences LSR Fortessa SORP analyzer w/HTS (7-laser excitation, 20-color detection, high-throughput sampler) -(2) BD Biosciences FACSVerse analyzer(s), (3-laser excitation, 8-color detection, universal high-throughput sampler multi-well plates or tubes, volumetric measurement for absolute cell counts) -Compucyte iCys laser scanning cytometer (3-laser excitation, 5-parameter detection), shared with NCI -Amnis ImageStreamX Mark II high resolution imaging cytometer (7-laser excitation, 3 imaging objectives, IdeasTM analysis software) In years past the FCS core has housed and utilized other cytometers (listed below) to support the NIAMS mission. These have since been retired and replaced with the more modern instruments listed above. -Beckman Coulter MoFlo high-speed cell sorter -Beckman Coulter CyAn ADP analyzer -BD Biosciences FACSCalibur -BD Biosciences FACS Vantage DiVa high-speed cell sorter -BD Biosciences LSR I 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 readily served by flow cytometry such as bone, muscle and skin biology. The facility also has the capability of sorting patient specimens and human-derived cells with a higher degree of safety. Following the recent addition of the FACSAria Fusion cell sorter, housed in a laminar flow biosafety cabinet, the facilitys existing BD InFlux cell sorter likewise gained its own biosafety cabinet during the core facility relocation in June 2014. These cell sorters now provide safer opportunities for NIAMS investigators to further the Bench to Bedside initiative promoted by the NIH Director, Dr. Francis Collins. Each equipped with seven lasers for excitation and 20 individual fluorescence detectors, both the Influx cell sorter and LSR Fortessa analyzer continue to provide state-of-the-art tools that enable 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, tissue fixed to slides, multi-well plates (iCys), or cells in suspension as in traditional flow cytometry (ImageStreamX). 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 ImageStreamX Mark II allows for higher throughput for more robust statistics, a characteristic of standard flow cytometers, while providing the image analysis advantage of a microscope-based system. The FCS has three dedicated personnel to support the needs 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 ImageStreamX 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 and is now a Staff Scientist. Having completed his doctoral degree and postdoctoral fellowship in the field of immunology, he joined the Core with substantial experience with flow cytometry. He subsequently completed his FACSAria Operator training at BD Biosciences in 2013 and is now the primary operator of the FACSAria IIIu. -Mr. James Simone, Section Leader, manages the day-to-day operations of the facility and 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 NIH 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 250 NIAMS flow cytometry users have been trained to date on the various cytometers available in the facility. 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 technical support and consultation. To further support the NIAMS mission, the Flow Cytometry Section continues to investigate new techniques and applications in cytometry for the use of its investigators.