Cell Biology and Imaging

细胞生物学和成像

• 批准号: 7217646
• 负责人: Joan Heller Brown
• 金额: $ 21万
• 依托单位: SAN DIEGO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217646
• 关键词: animal tissue; biomedical facility; cardiac myocytes; cell bank /registry; confocal scanning microscopy; data collection; fluorescence microscopy; fluorescence resonance energy transfer; genetically modified animals; information dissemination; laboratory mouse; laboratory rat; molecular /cellular imaging; tissue /cell culture; transfection /expression vector

The Cell Biology and Imaging Core (Core B) which will be used by all of the Projects will be involved in three primary functions: 1) preparation of neonatal rat ventricular myocytes (NRVMs) 2) preparation of adult mouse ventricular myocytes (AMVMs) 3) live cell imaging studies 4) distribution and analysis of results through use of the Open Microscopy Environment (OME) in coordination with The Scripps Research Institute (TSRI) The Cell Biology and Imaging Core (Core B) which will be used by all of the Projects will be involved in three primary functions: 1) preparation of neonatal rat ventricular myocytes (NRVMs), 2) preparation of adult mouse ventricular myocytes (AMVMs), and 3) live cell imaging studies. Consistent preparation of cardiomyocytes is critical to the ability to integrate findings across the Projects in the Program. Accordingly, while many of the individual laboratories have experience or capability in this regard, Core B will serve as a repository to insure cell availability and cross integration. Preparation of stable neonatal rat ventricular myocytes is relatively simple, but experimental observations are highly dependent on the methodology in particular the separation of cardiomyocytes from fibroblasts, plating density and choice of culture medium. With regard to adult mouse ventricular cardiomyocytes the mechanics and success of the preparation, and in particular the yield and long term viability of these cells can require skill and consistency. A dedicated technician and facility is therefore prerequisite to providing cells from TG and KO mice as required by virtually every project. Core C will also facilitate in the development of methods for expressing genes through adenoviral infection and expressing proteins through preparation and delivery of TAT fusion proteins. Live cell imaging will also be performed by Core B. In particular, technical advice and assistance with experiments using the FRET based Akt activity probe BKAR will be provided, as will analysis of mitochondrial membrane potential (TMRE) and integrity (RIRR, Calcein). Support for analysis of autophagy and mitophagy using TAT or adenoviral LC3 or cardiomyocytes from LC3 transgenics will also be provided. Inverted fluorescence and confocal microscopes equipped for these analyses are available at both UCSD and TSRI. The Open Microscopy Environment (OME) operated through TSRI provides a unique opportunity for data sharing and analysis.

所有项目将使用的细胞生物学和成像核心（核心B）将参与 三个主要功能： 1）新生大鼠心室肌细胞（NRVM）的制备 2）制备成年小鼠心室肌细胞（AMVM） 3）活细胞成像研究 4）通过使用开放显微镜环境（OME）分布和分析结果 与Scripps研究所（TSRI）的协调 所有项目将使用的细胞生物学和成像核（核心B）将参与三个 主要功能：1）新生大鼠心室心肌细胞（NRVM）的制备，2） 成年小鼠心室心肌（AMVM）和3）活细胞成像研究。一致的准备 心肌细胞对于整合计划中各个项目的发现的能力至关重要。因此， 尽管许多个人实验室在这方面都有经验或能力，但核心B将成为 确保细胞可用性和交叉积分的存储库。制备稳定的新生大鼠心室 肌细胞相对简单，但是实验观察高度依赖于该方法论 尤其是心肌细胞与成纤维细胞的分离，电镀密度和培养基的选择。 关于成年小鼠心室心肌细胞的制剂的力学和成功， 特别是这些细胞的产量和长期生存能力可能需要技巧和一致性。专用 因此 几乎每个项目。核心C还将促进通过通过 腺病毒感染和通过制备和递送TAT融合蛋白表达蛋白质。居住 细胞成像也将由CoreB进行。特别是技术建议和帮助 将提供使用基于FRET的AKT活动探针BKAR的实验，以及对 线粒体膜电势（TMRE）和完整性（RIRR，钙软件素）。支持自噬分析 还将提供使用TAT或腺病毒LC3或LC3转基因的心肌细胞的线粒体。 在两个UCSD上都可以使用倒荧光和共聚焦显微镜 和TSRI。通过TSRI操作的开放显微镜环境（OME）提供了独特的机会 用于数据共享和分析。