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的流式细胞仪核心为NEI室内社区提供流式细胞仪分析和分类设备和服务。它利用并开发了最新的样本准备，数据获取和分析以及协作研究项目中的分类程序。向学生，研究员和主要调查人员提供有关样本准备，染色和后处理后处理的培训。评估技术研究需求，并建议招募适当的员工，并收购满足这些需求所需的设备。今年，国家眼科研究所通过增加了新的分类器对生物安全进行了巨大的投资。将新的BD FACSARIA融合流式细胞仪添加到核心中。该分类器符合新的NIH的运营商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个不同实验室的六十三个人使用了该设施。这些服务和协作服务是为来自8个NEI Labs（ERPD，LI，LRCMB，MSF，NRL，RN，RN，RN，OGVFB和UNGIRD）的8位主要研究人员（PIS）的3个PIS进行的，加上来自NIH的其他3个Institutes的3个PI（NICHD，NIDCD和NIDDK）。分析了超过17,700个样品。超过20多个用户接受了如何操作MacSquant仪器的培训。今年，核心进行了大约1,400小时的分类。 在核心中使用的技术中，在核心中使用的方法是表型活细胞的方法，检测基因表达，监测膜和DNA含量因细胞凋亡或增殖而变化，测量细胞内蛋白质以及可溶性蛋白质的定量。 涉及人体组织的工作包括分类外周血单核细胞，以研究其细胞因子产生，基因型和DNA或RNA表达。来源是血液，巴菲大衣和白细胞。一些分析工作是用眼睛流体，眼组织标本，蛋白质和眼泪进行的。没有核心存储组织。 训练： 技术IRTA位置的可用性继续对核心操作产生非常积极的影响。训练有素的流式细胞仪操作员非常需要。通过TechIRTA，该设施的工作时间已从40小时/周扩大到60小时/周。扩大的工作时间有助于减少分类器时间表的积压。 TechIRTA位置大大提高了核心使用细胞分系程序进行高吞吐量多色分析的能力。 今年，核心主持了一名夏季学生，来自视觉研究和眼科多样性（Divro）暑期实习计划。该学生用自发葡萄膜炎的视网膜和小鼠的视网膜和淋巴组织中的IRBP特异性T细胞和浆细胞进行检测。 核心向NEI社区提供了几次正式的培训课程。这些课程包括：流式细胞仪概论，高级技术（例如，凋亡应用），分析仪器操作和FLOJO软件的数据分析。 核心经理完成了25小时的继续教育，以管理核心实验室的管理和8小时的核心设施管理培训。技术IRTA在研究所外完成了40个小时的正规培训。 核心并不义务用户承认其出版物中的核心贡献。以下是承认使用NEI流式细胞术核心资源的出版物的示例： Joe Mk1，Kwon HS，Cojocaru R，Tomarev SI。 肌动蛋白调节细胞增殖和生存。 J Biol Chem。 2014年4月4日； 289（14）：10155-67。 doi：10.1074/jbc.m113.547091。 EPUB 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.芳基烃受体AHR在小鼠中的缺失，降低了小鼠的含量下含微糖和RPE a traphy。投资Ophthalmol Vis Sci。 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. ER应力引起的通过分泌途径对错误折叠的GPI锚定蛋白的清除。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 Gao, Teresa A. Longo, Pinghu Liu, Kunio Nagashima, Rivka A. Rachel, Tiansen Li, Lijin Dong & Anand Swaroop.纤毛疗法相关的基因CC2D2A促进了纤毛生物发生期间母亲中心座的副阑尾组装。纳特社区。 2014年6月20日； 5：4207。 doi：10.1038/ncomms5207。 约翰逊电视台，西北Dekorver NW，Levasseur VA，Osborne A，Tassoni A，Lorber B，Heller JP，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由血小板衍生的生长因子赋予的视网膜神经节细胞神经保护。脑。 2014年2月; 137（PT 2）：503-19。 doi：10.1093/脑/AWT292。 Epub 2013 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 Autoimmun。 2014年5月； 50：12-22。 doi：10.1016/j.jaut.2013.08.003。 EPUB 2013年9月7日。 Horai R，Silver PB，Chen J，Agarwal RK，Chong WP，Jittayasothorn Y，Mattapallil MJ，Nguyen S，Natarajan K，Natarajan K，Villasmil R，Wang P，Karabekian Z，Lytton Z，Lytton SD，Chan CC，Caspi Rr。在表达特异性视网膜自身抗原特异性的转基因T细胞受体的小鼠中免疫特权和自发自身免疫的崩溃。 J Autoimmun。 2013年8月； 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。 基因表达在视网膜小胶质细胞中的变化：与小胶质细胞支持功能的关系和激活的调节。神经biol衰老。 2013年10月； 34（10）：2310-21。 doi：10.1016/j.neurobiolaging.2013.03.022。 EPUB 2013 APR 19。