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.

总结： 该细胞仪核心设施支持大多数NIAMS IRP研究人员的研究。此外，它还积极参与细胞仪培训，数据分析和流式细胞仪检测开发，为新用户提供这项技术。 该科目前存放和维护下列仪器，供国家原子能机构监测系统使用： - （1）BD Biosciences FACSAria Fusion细胞分选仪（5激光激发，19色检测，自动细胞沉积单元，生物安全柜） - （1）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） - （1）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） - （1）BD Biosciences FACSCanto SORP分析仪w/HTS（4激光激发，14色检测，高通量采样器） - （1）BD Biosciences LSR Fortessa SORP分析仪w/HTS（7激光激发，20色检测，高通量采样器） - （2）BD Biosciences FACSVerse分析仪，（3激光激发，8色检测，通用高通量取样器多孔板或管，绝对细胞计数的体积测量） -Compucyte iCys激光扫描细胞仪（3激光激发，5参数检测），与NCI共用 -Amnis ImageStreamX Mark II高分辨率成像细胞仪（7激光激发，3成像物镜，IdeasTM分析软件） 在过去的几年里，FCS核心已经容纳和使用了其他细胞仪（如下所列）来支持NIAMS使命。这些文书后来被淘汰，代之以上文所列的更现代的文书。 - 贝克曼库尔特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容易服务的学科，如骨骼、肌肉和皮肤生物学。 该设施还能够以更高的安全性分选患者标本和人源细胞。在最近增加了FACSAria Fusion细胞分选机（安装在层流生物安全柜中）之后，该设施现有的BD InFlux细胞分选机在2014年6月核心设施搬迁期间也获得了自己的生物安全柜。这些细胞分选仪现在为NIAMS研究人员提供了更安全的机会，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分析仪均配备了7个激发激光器和20个独立的荧光检测器，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有关细胞、固定在载玻片上的组织、多孔板（iCys）或悬浮细胞的荧光和形态学数据，如传统流式细胞术（ImageStreamX）中一样。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，ImageStreamX 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优势。 FCS有三名专职人员支持NIAMS IRP调查员的需求： Jeffrey Lay先生自2010年以来一直是该部门的一部分。他最近接受了使用Amnis 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，现在是一名科学家。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，在流式细胞术方面拥有丰富的经验。随后，他于2013年在BD Biosciences完成了FACSAria操作员培训，现在是FACSAria IIIu的主要操作员。 - James Simone先生，科长，管理该设施的日常运作，并继续参与在NIH和中大西洋地区推广流式细胞术。作为NIH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250多名NIAMS流式细胞仪用户接受了该设施现有各种细胞仪的培训。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。然而，在所有情况下，FCS工作人员都可以提供技术支持和咨询。 为了进一步支持NIAMS的使命，流式细胞术科继续研究细胞术的新技术和应用，供其研究人员使用。