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

流式细胞术支持免疫、皮肤、肌肉和骨骼疾病的研究

• 批准号: 9563188
• 负责人: Massimo Gadina
• 金额: $ 158.45万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9563188
• 关键词: Apoptosis; Autoimmune Diseases; Biological Assay; Biological Sciences; Biology; Bone Diseases; Cell Count; Cell Separation; Cell fusion; Cells; Characteristics; Computer software; Consultations; Contractor; Core Facility; Cytometry; Data; Data Analyses; Deposition; Detection; Development; Discipline; Disease; Doctor of Philosophy; Doctor' s Degree; Equipment; Fellowship; Flow Cytometry; Fluorescence; Fluorochrome; Functional disorder; Goals; Human; Human Resources; Hypersensitivity; Image; Image Analysis; Immune; Immune System Diseases; Immune System and Related Disorders; Immunology; Immunophenotyping; Individual; Instruction; Interest Group; International; Kinetics; Laboratories; Lasers; Measurement; Microscope; Mid-Atlantic Region; Mission; Modernization; Morphology; Muscle; Myopathy; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Patients; Policies; Proteins; Reporter; Research; Research Personnel; Research Support; Safety; Sampling; Scanning; Scientist; Services; Skin; Slide; Societies; Sorting - Cell Movement; Specimen; Speed; Suspensions; System; Techniques; Time; Tissues; Training; Tube; United States National Institutes of Health; Vendor; assay development; atopy; base; bench to bedside; bone; color detection; data acquisition; design; detector; experience; high resolution imaging; instrument; instrumentation; meetings; novel; operation; peer; skin disorder; statistics; tool

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.

概括： 该细胞仪核心设施支持大多数NIAMS IRP研究人员的研究。此外，它正在积极参与该技术新手的用户的细胞仪训练，数据分析和流式细胞仪测定开发。 该部分目前包含并维护以下NIAMS的仪器： - （1）BD Biosciences FACSARIA融合细胞分系器（5射线激励，19色检测，自动化细胞沉积单元，生物安全柜） - （1）BD Biosciences FACSARIA IIIU细胞分类器（5射线激励，19色检测，自动化细胞沉积单元） - （1）BD Biosciences涌入细胞分类器（7激光激发，20色检测，自动细胞沉积单元，光谱分析仪） - （1）BD Biosciences FACSCANTO SORP分析仪W/HTS（4射线激发，14色检测，高通量采样器） - （1）BD Biosciences LSR Fortessa Sorp分析仪W/HTS（7射线激励，20色检测，高通量采样器） - （2）BD Biosciences FACSVERSE分析仪（3射线激发，8色检测，通用高通量采样器多孔板或试管，绝对细胞计数的体积测量） - 与NCI共用 -AMNIS Imagestreamx Mark II高分辨率成像细胞仪（7射线激发，3个成像目标，IDEASTM分析软件） 在过去的几年中，FCS Core已经容纳并利用了其他细胞仪（下面列出）来支持NIAMS任务。此后，这些已退休并用上面列出的更现代的乐器取代。 -Beckman Coulter Moflo高速细胞分选仪 -Beckman Coulter Cyan ADP分析仪 -bd Biosciences FACSCALIBUR -BD Biosciences FACS Vantage Diva高速细胞分选仪 -bd Biosciences LSR I 除了关注免疫系统机制和功能障碍的实验室的项目外，该设施继续为NIAMS实验室提供流式细胞仪和分类服务，从而从事学科的实验室，无法通过诸如骨骼，肌肉和皮肤生物学等流式细胞仪仪式来提供学科。 该设施还具有更高安全程度的患者标本和人类衍生细胞的能力。 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. 每个配备了7个激发激光和20个单独的荧光探测器的激光器，涌入细胞分类器和LSR Fortessa分析仪都继续提供最先进的工具，使研究人员能够利用最新的荧光色素和荧光蛋白报道器。 那些研究人员涉及不太适合流式细胞术的标本的研究者可以利用该部分的激光扫描细胞仪，胸部（Compucyte）ICYS以及AMNIS Imagestreamx Mark II。这些基于显微镜的仪器提供了有关细胞，固定在幻灯片，多孔板（ICYS）或悬浮液中的细胞的荧光和形态数据，例如传统的流式细胞仪（ImagestReamx）。除了典型的免疫表型，凋亡测定和共定位研究外，ICYS还可以随着时间的流逝重新分析特定细胞的基于动力学的测定。相比之下，Imagestreamx Mark II的设计允许更高的吞吐量以获得更健壮的统计数据，这是标准流式细胞仪的特征，同时提供了基于显微镜系统的图像分析优势。 FCS有三名专用人员来支持NIAMS IRP调查人员的需求： -先生。杰弗里·莱（Jeffrey Lay）自2010年以来一直是该部分的一部分。他最近接受了使用Amnis Imagestreamx的培训，并将继续促进和支持该乐器的使用。 - 凯文·廷斯利（Kevin Tinsley）博士于2013年1月上旬以承包商的身份加入了核心设施，现在是一名员工科学家。在免疫学领域完成了博士学位和博士后研究金之后，他以流式细胞仪的丰富经验加入了核心。随后，他于2013年在BD Biosciences完成了FACSARIA运营商培训，现在是Facsaria IIIU的主要运营商。 -先生。部门负责人詹姆斯·西蒙妮（James Simone）管理该设施的日常运营，并继续参与NIH和中大西洋地区的流式细胞仪的促进。作为NIH流式细胞仪兴趣组的联合主席，他协助组织和促进季度会议，以展示该领域的领先研究人员，并促进与流式细胞仪相关产品的供应商的关系。他在国际细胞仪（ISAC）和ISAC生物安全委员会的长期成员资格有助于促进与来自世界各地的同伴交流新想法和信息。 迄今为止，已在设施中可用的各种细胞仪上对250多个NIAM流式细胞仪使用者进行了培训。所有新用户都参加了对设施设备和政策的一般介绍和方向。然后，根据需求和以前的经验，他们会发展到最适合其特定研究需求的工具的个性化或小组指导。经过该部门负责人的批准后，用户可以无助地操作工具。但是，在所有情况下，FCS工作人员都可以进行技术支持和咨询。 为了进一步支持NIAMS的任务，流式细胞仪部分继续研究细胞仪中使用其研究人员的新技术和应用。