CORE--CELL BIOLOGY SUPPORT

自1978年成立以来，该核心为研究人员提供了 各种特性良好的培养细胞的计划项目 来自血管和非血管来源。核心人员也有 与研究人员密切合作，根据需要开发新的培养系统 以促进该计划的科学目标。在上一个项目中 在此期间，这包括开发了两种新的方法 用流式细胞仪分选和抗体分离小鼠肺内皮细胞 成对的磁珠。目前正在进行工作，以扩大 这些方法在分离小鼠内皮细胞中的应用 来自不同的微血管床，包括心脏、肾脏和大脑。 在下一个项目期间，这将是一个主要优先事项，并应 允许研究人员检查这些之间潜在的重要差异 正常和遗传性分离的各种类型的内皮细胞 经过改良的动物。 核心单位A将继续努力尽量减少成本重复 在本计划内并提供关键的质量控制测试 生物试剂。胎牛血清，生长和附着因子， 和酶由核心人员定期测试其有效性，以及 然后批量购买，供项目研究人员使用。这 确保在核心中衍生的细胞培养在AS中保持 从他们最初的孤立到现在尽可能保持一致 它们在项目中的实验用途。核心将继续提供 一般细胞培养方法和无菌技术方面的培训 项目人员(例如，技术人员、学生、研究员)。堆芯 人员还将培训新的人员在技术和程序方面 对于安全处理、净化和处置垃圾是必要的 生物危险材料，如人体组织、人类血液和病毒 被感染的细胞。 在下一个项目期间，Core I将承担两项新职能。 第一，维持胚胎干细胞系和额外的细胞 将携带小鼠“基因打靶”所需的培养材料 由核心A级人员出动。在之前的项目期内， 转基因/ES细胞核心产生的小鼠携带几个新的突变 内皮细胞黏附分子，从而获得专业知识和 证明了在基因操作方面的已被证明的记录 小鼠在体外和体内的基因组(1-3)。在续约期内， 这一领域的专业知识将被纳入细胞生物学核心。AS 如下所述，核心A人员将协助维护ES 细胞系，而实际的囊胚注射将在 LMRC机构转基因核心设施(在 阿琳·夏普医生。第二，核心将维持和监督 一种新的荧光显微镜/计算机图像分析系统和 现有的荧光分光光度/显微系统将是 被项目1、2、3和4大量使用。新的计算机映像 系统将使项目研究人员能够访问最先进的 定量荧光显微镜、图像分析和出版 高质量的彩色和B/2数码打印。现有的SPEX双励磁 分光光度计将允许常规荧光分析和寿命 细胞内钙离子浓度的荧光显微镜分析 使用Fura-2和新一代非紫外线荧光探针 胞浆中一价或二价阳离子和pH值的测定。Dr。 卢辛斯卡斯和爱德华·马库斯先生将提供协助 研究人员计划和执行这样的实验。

Since its inception in 1978, this core has provided researchers within the Program Project with a wide variety of well-characterized cultured cells from both vascular and non-vascular sources. Core personnel also have worked closely with researchers to develop new culture systems, as needed to facilitate the scientific goals of the Program. During the last project period, this has included the development of two new methodologies for the isolation of mouse lung endothelium using FACS sorting and antibody- coupled magnetic beads. Work is currently underway to expand the application of these methodologies to isolate murine endothelial cells from different microvascular beds, including the heart, kidney and brain. During the next project period, this will be a major priority, and should allow researchers to examine potentially important differences among these various types of endothelium isolated from both normal and genetically modified animals. Core Unit A will continue in its efforts to minimize cost duplication within the Program and to provide quality control testing of key biological reagents. Fetal bovine serum, growth and attachment factors, and enzymes are routinely tested for their efficacy by Core personnel, and then purchased in bulk quantities for use by Program researchers. This ensures that cell cultures derived in the Core are maintained in as consistent a fashion as possible from their initial isolated through to their experimental uses in the Projects. The Core will continue to provide training in general cell culture methods and in sterile technique to new Program personnel (e.g., technicians, students, research fellows). Core personnel also will train new personnel in the techniques and procedures necessary for the safe handling, decontamination, and disposal of biohazardous materials such as human tissue, human blood, and virally infected cells. During the next project period, Core I will assume two new functions. First, the maintenance of embryonic stem cell lines and additional cell culture materials required for "gene targeting" in mice will be carried out by Core A personnel. During the previous project period, the Transgenic/ES Cell Core produced mice bearing several new mutations in endothelial cell adhesion molecules, thus gaining expertise and demonstrating a proven track record in the genetic manipulation of the murine genome both in vitro and in vivo (1-3). In the renewal period, expertise in this area will be incorporated into the Cell Biology Core. As described below, Core A personnel will assist in the maintenance of ES cell lines, while the actual blastocyst injections will be carried out in the LMRC Institutional Transgenic Core facility (under the supervision of Dr. Arlene Sharpe. Second, the Core will maintain and oversee the use of a new fluorescence microscopy/computerized image analysis system and an existing fluorescent spectrophotometric/microscopy system which will be heavily utilized by Projects 1, 2, 3, and 4. The new computer imaging system will give Project researchers access to state-of-the-art quantitative fluorescence microscopy, image analysis and publication quality color and B/2 digital printing. The existing SPEX dual excitation spectrophotometer will allow general fluorimetry and live time fluorescence microscopic analysis of intracellular Ca2+ concentrations using Fura-2 and newer generation non-UV fluorescent probes for measurement of cytosolic monovalent or divalent cations and pH. Dr. Luscinskas and Mr. Edward Marcus will be made available to assist researchers in planning and executing such experiments.