Flow & Imaging Cytometry Core Facility

NINDS流动和成像细胞术核心设施(设施)由NINDS DIR于2001年建立，是NINDS和其他内部研究人员支持其基础和临床研究计划工作的重要资源。该设施不断提供对该设施管理的流式细胞仪和成像设备的无缝使用，并提供关键的专门知识，以培训调查人员正确和最佳地使用这种设备。该设施的工作人员还可以为设计和优化各种新颖和定制的多重染色方案提供建设性的意见，使用适当的细胞表面、细胞质、核、蛋白质、基因表达和生理指示剂染料生物标记物的独特组合，这些生物标志物是任何给定项目的发起人(S)特别感兴趣的。当需要时，设施工作人员例行进行多色流式细胞术和原位细胞术实验，并分析来自感兴趣的单个或多个细胞群体的数据。根据测试的生物标志物的不同组合，应用用户定义的布尔逻辑门控策略，该设备提供每个研究人员感兴趣的单个细胞或细胞群体中复杂信号分布的定量描述和解释。利用流式细胞仪的预备性细胞分选能力，设备工作人员可应发起调查人员的要求，协助从异种细胞制剂中分离高度纯化的细胞群体和亚群体。这包括从CNS和PNS组织中分选神经干细胞和前体细胞、特定的神经元和神经胶质细胞类型以及非神经细胞类型(小胶质细胞、内皮细胞)。此外，该机构还定期从骨髓、外周血、脾和脑脊液中分选非中枢神经系统来源的干细胞(造血系、间充质系和Hoechst染料除外)和不同的白细胞表型(淋巴细胞、单核细胞、中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞和肥大细胞)。这些高纯度的目标细胞随后被NINDS和其他壁内研究人员常规用于进一步的体外和体内研究，包括细胞培养、移植、延时和生理指示剂染料成像，以及转导或转染特定的目标基因结构。此外，FACS纯化的细胞已被用作蛋白质的宝贵来源，用于Western blotting和免疫沉淀实验，以及用于RT-PCR、微阵列、RNA-Seq和microRNA分析的RNA来源。这些细胞也为使用反义和siRNA探针进行特定的功能丧失实验提供了前所未有的见解。在上述应用中使用FACS纯化的同质细胞的主要意义在于，这种方法减少了研究异种细胞制剂时固有的发现的模糊性。总而言之，该设施提供的定量流动和原位细胞分析的常规应用以及预备性细胞分选服务为NINDS和其他内部调查人员提供了大量关于细胞生物学的见解，否则无法证明这一点。自2001年成立以来，该机构已经为500多名NINDS和其他内部调查人员、研究员、学生和科学工作人员提供了这些宝贵的服务，并支持了来自15个NIH研究所和中心的105个实验室的700多个基础、翻译和临床研究项目的多学科科学研究，最终在备受好评的生物医学和生物科学期刊上发表了240多篇同行评议的文章。在过去的12个月里，该基金支持了122名研究人员、研究员、学生和实验室工作人员的研究需求，为30个NINDS和其他内部实验室的125个基础科学、临床和转化研究项目做出了贡献，并促进了NINDS和其他6个NIH研究所和中心之间的多学科合作项目。在过去12个月中，这一机制的贡献被纳入了备受赞誉的科学期刊上14篇同行评审文章的出版物中，并加快了目前正在准备提交期刊或在期刊同行评审中的另外21篇文章的编写工作。

NINDS Flow and Imaging Cytometry Core Facility (Facility) was established in 2001 by NINDS DIR as a crucial resource to NINDS and other intramural investigators to support their work in both basic and clinical research programs. The Facility continuously provides seamless use of flow cytometry and imaging equipment managed by the Facility as well as the crucial expertise to train investigators in proper and optimal use of this equipment. The Facility staff is also available to provide constructive input in designing and optimization of a variety of novel and customized multiplex staining protocols using unique combinations of appropriate cell surface, cytoplasmic, nuclear, protein, gene expression and physiological indicator dye biomarkers that are of specific interest to initiating investigator(s) for any given project. When requested, the Facility staff routinely carry out multi-color flow and in situ cytometry experiments and analyze the data from single or multiple cell populations of interest. Applying user-defined Boolean logic gating strategies based on different combinations of biomarkers tested, the Facility provides quantitative accounts and interpretations of the complex signal distributions in individual cells or cell populations of interest to each investigator. Using the preparative cell sorting capability of flow cytometers, the Facility staff is available to assists with isolation of highly purified cell populations and subpopulations from heterogeneous cell preparations, as requested by the initiating investigator. These include sorting of neural stem and progenitor cells, specific neuronal and glial cell types, as well as non-neural cell types (microglia, endothelial cells) from both CNS and PNS tissues. In addition, the Facility routinely sorts non-CNS-derived stem cells (hematopoietic, mesenchymal and Hoechst dye-excluding side populations) and different leukocyte phenotypes (lymphocytes, monocytes, neutrophils, basophils, eosinophils, mast cells) from the bone marrow, peripheral blood, spleen, and cerebrospinal fluid. These highly-purified cells of interest are then routinely used by NINDS and other intramural investigators for further in vitro and in vivo studies including cell culture, transplantation, time-lapse and physiological indicator dye imaging, and for transduction or transfection with specific gene constructs of interest. Additionally, FACS-purified cells have been utilized as an invaluable source of proteins for Western blotting and immunoprecipitation experiments as well as a source of RNA for RT-PCR, microarray, RNA-Seq, and microRNA assays. These cells have also provided unprecedented insights into specific loss-of-function experiments using antisense and siRNA probes. The major significance in using FACS-purified homogeneous cells in the abovementioned applications is that this approach reduces the ambiguity in the findings inherently present when studying heterogeneous cell preparations. Taken together, the routine applications of quantitative flow and in situ cytometry assays and preparative cell sorting services provided by this Facility has produced numerous insights into cell biology for NINDS and other intramural investigators, which could not otherwise be demonstrated. Since inception in 2001, the Facility has provided these invaluable services to more than 500 NINDS and other intramural investigators, fellows, students, and scientific staff and supported the multidisciplinary scientific research in over 700 basic, translational, and clinical research projects from 105 laboratories across 15 NIH institutes and centers, culminating in publication of over 240 peer-reviewed articles in highly acclaimed biomedical and biological scientific journals. Over the past 12 months, the Facility supported the research needs of 122 investigators, fellows, students, and laboratory staff contributing to 125 basic science, clinical and translational research projects in 30 NINDS and other intramural laboratories and facilitated multidisciplinary collaborative projects between NINDS and 6 other NIH institutes and centers. Contributions by this Facility over the past 12 months were incorporated in publications of 14 peer-reviewed articles in highly acclaimed scientific journals and expedited the preparation of 21 more articles currently in preparation for journal submission or in journal peer review.