Flow Cytometry CORE

流式细胞术核心

• 批准号: 8938502
• 负责人: Rachel R. Caspi
• 金额: $ 26.88万
• 依托单位: NATIONAL EYE INSTITUTE
• 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/8938502
• 关键词: 2 year old; 5 year old; 7 year old; Age; Aging; Apoptosis; Aryl Hydrocarbon Receptor; Atrophic; Autoantigens; Autoimmunity; Blood; Brain; Budgets; CNS autoimmunity; Cell Proliferation; Cell Survival; Cells; Centrioles; Communities; Computer software; Continuing Education; Core Facility; DNA; Data Analyses; Detection; Equipment; Eye; Flow Cytometry; GRB10 gene; Gene Expression; Gene Expression Profiling; Genes; Genetic Transcription; Genotype; Hour; Immune; Individual; Institutes; Internships; Investments; Laboratories; Leukocytes; Life; Liquid substance; Lymphoid Tissue; MDK gene; Measurement; Membrane; Mesenchymal Stem Cells; Methods; Microglia; Mothers; Mus; National Eye Institute; National Institute of Child Health and Human Development; National Institute of Diabetes and Digestive and Kidney Diseases; National Institute on Deafness and Other Communication Disorders; Ophthalmology; Pathway interactions; Peripheral Blood Mononuclear Cell; Phenotype; Plasma Cells; Platelet-Derived Growth Factor; Ploidies; Positioning Attribute; Preparation; Principal Investigator; Procedures; Production; Proteins; Publications; Regulation; Research; Research Project Grants; Resources; Retina; Retinal; Retinal Ganglion Cells; Sampling; Schedule; Services; Silver; Sorting - Cell Movement; Source; Specimen; Staining method; Stains; Stress; Students; T-Cell Receptor; T-Lymphocyte; Techniques; Tissues; Training; Transgenic Organisms; United States National Institutes of Health; Uveitis; Vision research; Work; Yang; appendage; ciliopathy; cilium biogenesis; cytokine; data acquisition; human tissue; instrument; instrumentation; meetings; myocilin; neuroprotection; operation; programs; response; square foot; tear proteins

The Flow Cytometry Core at NEI provides flow cytometry analytical and sorting equipment and services to the NEI Intramural community. It utilizes and develops state-of-the-art sample preparation, data acquisition and analysis, and sorting procedures in collaborative research projects. Provides training to students, fellows, and principal investigators on sample preparation, staining, and post-sort handling. Assesses technical research needs and recommends recruitment of the appropriate staff and acquisition of the equipment needed to meet those needs. This year the National Eye Institute made a great investment in biosafety with the addition of a new sorter. A new BD FACSAria Fusion flow cytometer with a fully integrated biosafety cabinet was added to the core. This sorter meets the new NIH's operator and sample protection requirements as well as global standards for bioprotection. OPERATION OF THE CORE: This year, sixty-three individuals from thirteen different laboratories used the facility. These services and collaborative services were performed for 8 Principal Investigators (PIs) from 8 NEI labs (ERPD, LI, LRCMB, MSF, N-NRL, RN, OGVFB and UNGIRD), plus 3 PIs from 3 other institutes at NIH (NICHD, NIDCD and NIDDK). Over 17,700 samples were analyzed. More than twenty users received training how to operate the MACSQuant instrumentation. This year the core performed about 1,400 hours of sorting. Among the techniques now in use within the core are methods for phenotyping live cells, detecting gene expression, monitoring membrane and DNA content changes due to apoptosis or proliferation, measurement of intracellular proteins and quantification of soluble proteins. The work involving human tissues includes the sorting of peripheral blood mononuclear cells to study their cytokine production, genotype and DNA or RNA expression. The sources are blood, buffy coat, and white cells. Some analytical work had been done with eye fluids, eye tissue specimen, protein, and tears. No tissues were stored by the core. TRAINING: The availability of the Technical IRTA position continues to have a very positive effect on the Core operation. There is a great need for well-trained flow cytometry operators. Through the TechIRTA, the hours of operation of the facility have been expanded from 40 hr/week to 60 hr/week. The expanded hours of operation helped to reduce the backlog on the sorter schedule. The TechIRTA position has greatly enhanced the ability of the Core to use the cell sorter for high throughput multicolor analysis. This year the core hosted a summer student from the Diversity In Vision Research and Ophthalmology (DIVRO) summer internship program. The student worked in detection of IRBP specific T cells and plasma cells in retina and lymphoid tissue of mice with spontaneous uveitis. Several formal training sessions were offered by the core to the NEI community. These courses included: Introduction to Flow Cytometry, Advanced Techniques (Ex. Apoptosis applications), Analytical Instrument Operation and Data Analysis with FloJo Software. The Core Manager completed 25 hours of continuing education in management of core laboratories and 8 hours of training in management of Core facilities. The technical IRTA completed 40 hours of formal training outside the Institute. The Core does not obligate users to acknowledge the Core contribution in their publications. The following are examples of the publications that acknowledged the use of NEI Flow Cytometry Core resources: Joe MK1, Kwon HS, Cojocaru R, Tomarev SI. Myocilin regulates cell proliferation and survival. J Biol Chem. 2014 Apr 4;289(14):10155-67. doi: 10.1074/jbc.M113.547091. Epub 2014 Feb 22. Kim SY, Yang HJ, Chang YS, Kim JW, Brooks M, Chew EY, Wong WT, Fariss R, Rachel R, Cogliati T8, Qian H, Swaroop A. Deletion of aryl hydrocarbon receptor AHR in mice leads to subretinal accumulation of microglia and RPE atrophy. Invest Ophthalmol Vis Sci. 2014 Aug 26. pii: IOVS-14-15091. doi: 10.1167/iovs.14-15091. Satpute-Krishnan P, Ajinkya M, Bhat S, Itakura E, Hegde RS, Lippincott-Schwartz J. ER Stress-Induced Clearance of Misfolded GPI-Anchored Proteins via the Secretory Pathway. Cell. 2014 Jul 31;158(3):522-33. doi: 10.1016/j.cell.2014.06.026. Shobi Veleri, Souparnika H. Manjunath, Robert N. Fariss, Helen May-Simera, Matthew Brooks, Trevor A. Foskett, Chun Gao, Teresa A. Longo, Pinghu Liu, Kunio Nagashima, Rivka A. Rachel, Tiansen Li, Lijin Dong & Anand Swaroop. Ciliopathy-associated gene Cc2d2a promotes assembly of subdistal appendages on the mother centriole during cilia biogenesis. Nat Commun. 2014 Jun 20;5:4207. doi: 10.1038/ncomms5207. Johnson TV, DeKorver NW, Levasseur VA, Osborne A, Tassoni A, Lorber B, Heller JP, Villasmil R, Bull ND, Martin KR, Tomarev SI. Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome. Brain. 2014 Feb;137(Pt 2):503-19. doi: 10.1093/brain/awt292. Epub 2013 Oct 30. Chong WP, Horai R, Mattapallil MJ, Silver PB, Chen J, Zhou R, Sergeev Y, Villasmil R, Chan CC, Caspi RR. IL-27p28 inhibits central nervous system autoimmunity by concurrently antagonizing Th1 and Th17 responses. J Autoimmun. 2014 May;50:12-22. doi: 10.1016/j.jaut.2013.08.003. Epub 2013 Sep 7. Horai R, Silver PB, Chen J, Agarwal RK, Chong WP, Jittayasothorn Y, Mattapallil MJ, Nguyen S, Natarajan K, Villasmil R, Wang P, Karabekian Z, Lytton SD, Chan CC, Caspi RR. Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen. J Autoimmun. 2013 Aug;44:21-33. doi: 10.1016/j.jaut.2013.06.003. Epub 2013 Jun 28. Ma W, Cojocaru R, Gotoh N, Gieser L, Villasmil R, Cogliati T, Swaroop A, Wong WT. Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation. Neurobiol Aging. 2013 Oct;34(10):2310-21. doi: 10.1016/j.neurobiolaging.2013.03.022. Epub 2013 Apr 19.

NEI 的流式细胞术核心为 NEI 校内社区提供流式细胞术分析和分选设备及服务。它在合作研究项目中利用和开发最先进的样品制备、数据采集和分析以及分类程序。为学生、研究员和主要研究人员提供样品制备、染色和分选后处理方面的培训。评估技术研究需求并建议招聘适当的人员并采购满足这些需求所需的设备。今年，国家眼科研究所在生物安全方面进行了大量投资，增加了一台新的分选机。核心中添加了带有完全集成生物安全柜的新型 BD FACSAria Fusion 流式细胞仪。该分选机满足新的 NIH 操作员和样品保护要求以及全球生物保护标准。 核心的操作： 今年，来自 13 个不同实验室的 63 名个人使用了该设施。这些服务和协作服务是为来自 8 个 NEI 实验室（ERPD、LI、LRCMB、MSF、N-NRL、RN、OGVFB 和 UNGIRD）的 8 位首席研究员 (PI) 以及来自 NIH 其他 3 个研究所（NICHD、 NIDCD 和 NIDDK）。分析了超过 17,700 个样本。二十多名用户接受了如何操作 MACSQuant 仪器的培训。今年，核心执行了约 1,400 小时的分拣工作。 目前核心使用的技术包括对活细胞进行表型分析、检测基因表达、监测由于细胞凋亡或增殖导致的膜和 DNA 含量变化、测量细胞内蛋白质以及定量可溶性蛋白质的方法。 涉及人体组织的工作包括分选外周血单核细胞，以研究其细胞因子的产生、基因型以及 DNA 或 RNA 表达。来源是血液、血沉棕黄层和白细胞。对眼液、眼组织样本、蛋白质和眼泪进行了一些分析工作。核心没有储存任何组织。 训练： IRTA 技术职位的可用性继续对核心运营产生非常积极的影响。非常需要训练有素的流式细胞术操作员。通过 TechIRTA，该设施的运行时间已从 40 小时/周延长至 60 小时/周。延长的运营时间有助于减少分拣机日程上的积压。 TechIRTA 职位大大增强了 Core 使用细胞分选仪进行高通量多色分析的能力。 今年，该中心接待了一名来自视觉研究和眼科多样性 (DIVRO) 暑期实习项目的暑期学生。该学生致力于检测患有自发性葡萄膜炎的小鼠的视网膜和淋巴组织中的 IRBP 特异性 T 细胞和浆细胞。 核心人员向 NEI 社区提供了几次正式培训课程。这些课程包括：流式细胞术简介、先进技术（例如细胞凋亡应用）、分析仪器操作和使用 FloJo 软件进行数据分析。 核心经理完成了25小时的核心实验室管理继续教育和8小时的核心设施管理培训。 IRTA 技术人员在学院外完成了 40 个小时的正式培训。 Core 并不要求用户在其出版物中承认 Core 的贡献。以下是承认使用 NEI 流式细胞术核心资源的出版物示例： 乔 MK1、权 HS、科乔卡鲁 R、托马列夫 SI。 肌纤蛋白调节细胞增殖和存活。生物化学杂志。 2014 年 4 月 4 日；289(14)：10155-67。 doi：10.1074/jbc.M113.547091。电子版 2014 年 2 月 22 日。 Kim SY，Yang HJ，Chang YS，Kim JW，Brooks M，Chew EY，Wong WT，Fariss R，Rachel R，Cogliati T8，Qian H，Swaroop A。小鼠芳烃受体 AHR 的缺失导致小胶质细胞视网膜下积聚和 RPE 萎缩。投资眼科可见科学。 2014 年 8 月 26 日。pii：IOVS-14-15091。 doi：10.1167/iovs.14-15091。 Satpute-Krishnan P、Ajinkya M、Bhat S、Itakura E、Hegde RS、Lippincott-Schwartz J。 内质网应激诱导通过分泌途径清除错误折叠的 GPI 锚定蛋白。细胞。 2014 年 7 月 31 日；158(3):522-33。 doi：10.1016/j.cell.2014.06.026。 Shobi Veleri、Souparnika H. Manjunath、Robert N. Fariss、Helen May-Simera、Matthew Brooks、Trevor A. Foskett、Chun Gau、Teresa A. Longo、Pinghu Liu、Kunio Nagashima、Rivka A. Rachel、Tiansen Li、Lijin Dong ＆阿南德·斯瓦鲁普。纤毛病相关基因 Cc2d2a 在纤毛生物发生过程中促进母中心粒上远端附属物的组装。纳特·康姆. 2014 年 6 月 20 日；5:4207。 doi：10.1038/ncomms5207。 Johnson TV、DeKorver NW、Levasseur VA、Osborne A、Tassoni A、Lorber B、Heller JP、Villasmil R、Bull ND、Martin KR、Tomarev SI。通过分析间充质干细胞分泌组来鉴定血小板衍生生长因子赋予的视网膜神经节细胞神经保护作用。脑。 2014 年 2 月；137（第 2 部分）：503-19。 doi：10.1093/brain/awt292。电子版 2013 年 10 月 30 日。 Chong WP、Horai R、Mattapallil MJ、Silver PB、Chen J、Zhou R、Sergeev Y、Villasmil R、Chan CC、Caspi RR。 IL-27p28 通过同时拮抗 Th1 和 Th17 反应来抑制中枢神经系统自身免疫。 J自身免疫。 2014 年 5 月；50:12-22。 doi：10.1016/j.jaut.2013.08.003。电子版 2013 年 9 月 7 日。 Horai R、Silver PB、Chen J、Agarwal RK、Chong WP、Jittayasothorn Y、Mattapallil MJ、Nguyen S、Natarajan K、Villasmil R、Wang P、Karabekian Z、Lytton SD、Chan CC、Caspi RR。表达视网膜自身抗原特异性转基因 T 细胞受体的小鼠的免疫特权和自发性自身免疫的破坏。 J自身免疫。 2013 年 8 月；44:21-33。 doi：10.1016/j.jaut.2013.06.003。电子版 2013 年 6 月 28 日。 Ma W、Cojocaru R、Gotoh N、Gieser L、Villasmil R、Cogliati T、Swaroop A、Wong WT。 衰老视网膜小胶质细胞的基因表达变化：与小胶质细胞支持功能和激活调节的关系。神经生物学老化。 2013 年 10 月；34(10):2310-21。 doi：10.1016/j.neurobiolaging.2013.03.022。电子版 2013 年 4 月 19 日。