High-speed Flow Cytometer

申请者描述(申请人提供)：申请共享仪器赠款(SIG)购买一台新的流式细胞仪在儿童医院洛杉矶(CHLA)的遗传，免疫和干细胞治疗(GIST)计划。GIST计划由6名由美国国立卫生研究院资助的主要研究人员组成，他们进行基础研究、翻译研究和临床研究。超过25名博士后研究员、研究生和技术人员进行的研究严重依赖FACS进行细胞分析和分离。由于流式细胞术一直是GIST研究人员进行研究的关键方法学工具，该计划在过去12年中建立了自己的FACS核心实验室来支持其研究。目前的核心包括两个分析细胞仪和一个分选器，它们被安置在一个专门的FACS实验室中，并由2名FTE细胞仪技术人员提供支持。进行的基因转移研究包括从两种临床前模型(小鼠、嵌合鼠/人和狗模型)向细胞中转移基因的分析，以及对参与临床基因转移试验的患者的细胞进行分析和分类。临床前研究依赖于基于单抗的多参数流式细胞术和基于增强型绿色荧光蛋白(EGFP)的转导细胞标记。未来的研究有望越来越多地采用竞争性再繁殖分析，其中包含不同EGFP变体的载体将被用于标记具有不同载体设计或在不同条件下转导的细胞。该项目的免疫学研究主要集中在淋巴细胞的正常个体发育、造血干细胞移植(HSCT)后淋巴细胞的发育以及细胞因子受体在淋巴细胞生成中的作用。同样，这些研究依赖于基于抗体和基于绿色荧光蛋白的联合分析以及核酸染色来测量增殖和/或凋亡。受体功能的分析越来越多地集中在使用EGFP光谱变体和受体的融合蛋白的技术上；这些研究依赖于FACS分离细胞，使用广泛的颜色光谱来激发和发射。该计划成员进行的干细胞研究是对免疫表型典型的造血祖细胞以及越来越多的包括间充质细胞和上皮细胞在内的其他血统的祖细胞的基础生物学研究。干细胞研究还依赖于抗体、EGFP和核酸染色的多参数研究，以及对嵌合小鼠体内发育的后代以及体外培养的复杂分析。目前的拨款是购买一台新的细胞分选机，以提高GIST计划的生产率。分拣机将提供现有设备无法支持的额外分析和分拣能力。新的分选器还将通过为越来越多的GIST项目调查人员提供额外的分选时间来允许该计划的增长，从而防止FACS的可用性成为研究瓶颈。新的分选机将配置为最大的光谱范围，细胞分选的灵活性和速度，以及分选细胞的无菌。为了容纳新的分拣机，FACS核心实验室将用机构资金进行改建和扩建。将综合使用机构支助和赠款支助来配置和维护新设备。

DESCRIPTION (provided by applicant): The application is for a Shared Instrument Grant (SIG) to purchase a new flow cytometer for the Genetic, Immunologic and Stem Cell Therapy (GIST) program at Childrens Hospital Los Angeles (CHLA). The GIST program consists of 6 NIH-funded principal investigators who perform basic, translational, and clinical research. Over 25 post-doctoral fellows, graduate students, and technicians perform studies that are heavily dependent on FACS for analyses and isolation of cells. Because flow cytometry has been a key methodologic tool for the research performed by the GIST investigators, the program has built its own FACS Core Laboratory over the last 12 years to support its research. The present Core includes two analytic cytometers and one sorter, which are housed in a dedicated FACS laboratory and supported by 2 FTE cytometry technicians. Gene transfer studies that are performed include analyses of gene transfer into cells from both preclinical models (murine, chimeric mouse/human, and canine models) as well as analyses and sorting of cells from patients participating in clinical gene transfer trials. The preclinical studies depend on multiparametric flow cytometry with both monoclonal antibody-based and enhanced green fluorescent protein (EGFP)- based labeling of transduced cells. Future studies are expected to increasingly employ competitive repopulation assays in which vectors containing different EGFP variants will be used to mark cells transduced with different vector designs or under different conditions. Immunoloqic studies performed in the program are mainly focused on studies of the normal ontogeny of lymphocytes, lymphocyte development after hematopoietic stem cell transplantation (HSCT), and the function of cytokine receptors in lymphopoiesis. Again, these studies depend on combined antibody and EGFP-based assays as well as nucleic acid staining to measure proliferation and/or apoptosis. Analyses of receptor function are increasingly focused on techniques that employ fusion proteins of EGFP spectral variants and receptors; these studies depend on FACS isolation of cells using a broad spectrum of colors for excitation and emission. The stem cell studies performed by members of the program are basic biologic studies of immunophenotypically characterized hematopoietic progenitors, and increasingly, progenitors of other lineages including mesenchymal and epithelial cells. The stem cell studies also depend on multiparametric studies with antibodies, EGFP, and nucleic acid staining, and complex analyses of progeny that develop in vivo in chimeric mice as well as in vitro cultures. The present grant is to purchase a new cell sorter to increase the GIST program's productivity. The sorter will provide additional analytic and sorting capabilities, which the present equipment cannot support. The new sorter will also allow the program to grow by providing additional sort time for the increasing number of GIST program investigators, thus preventing FACS availability from becoming a research bottleneck. The new sorter will be configured for maximal spectral range, flexibility and speed of cell sorting, and sterility of sorted cells. In order to accommodate the new sorter, the FACS Core Laboratory will be remodeled and expanded with institutional funds. Combined institutional and grant support will be used to staff and maintain the new equipment.