Cell Isolation, Culture and Protein Expression Core

1.标题：细胞分离、培养及蛋白表达核心 2.核心单位负责人：Paul，DiCorleto，博士，克利夫兰诊所勒纳研究所所长 技术人员：张晓伦、Alona Merkulova和Angela Money。 3.说明 这一核心的目的是为该方案的四个项目提供所需的细胞和蛋白质。Core B人员将分离的特定细胞是：1)血细胞，主要是人类和小鼠来源的血小板；2)人和小鼠来源的内皮细胞；以及3)表达某些整合素、细胞骨架蛋白和这些蛋白的突变体的真核或原核细胞。原核细胞被用作纯化蛋白质的来源；Talin、kindlins和SCR家族激酶衍生物，在核心中，并根据需要提供给各个项目。由经验丰富的人员准备细胞将有助于确保纯度和功能的一致性。拥有项目所需的可靠的功能性血小板制剂来源特别有价值，因为血小板的功能可能是高度可变的。小鼠血管内皮细胞也是如此 实验进展通常依赖于使用从少数有价值的小鼠品系中提取的细胞进行实验的起源。纯化的蛋白质用于结合研究和结构分析，其中需要相对大量的高质量准备。由于需要制备数量有限的蛋白质，分离程序可以标准化，以提供高质量的产品。集中开展这些活动将节省资源，有助于确保质量，并允许对结果进行项目间比较。此外，由于个别项目仍将需要大量独立的细胞培养，并且无疑将需要为特定的实验分离细胞，核心将在适当的细胞培养和分离方法以及以适当的方式评估细胞质量方面对计划项目实验室的成员进行培训。因此，核心也起到了教育作用。 在之前的资助期间，细胞培养和表达是两个独立的实体。它们现在被整合成一个单一的核心。这一合并使新的核心能够向所有四个项目提供更公平的服务。以前，项目2很少使用细胞培养服务，项目3和4没有使用蛋白质表达核心。重要的是，合并还使我们能够将年度直接费用总额从以前的核心B+C的250 426美元减少到新的核心B的136 616美元。这种削减是必要的，因为年度预算撤销是必要的，而且是可行的，因为核心的活动是在邻近空间进行的，使核心人员能够分担责任。此外，通过节省预算，我们能够创建一个新的In Vivo Core C(111,900美元)，它提供了将在该计划中广泛使用的重要服务。 保罗·迪科莱托博士担任核心主任。DiCorleto博士指导了我们的细胞培养核心 计划，该计划之前向计划项目提供外周血细胞和内皮细胞。他是 有资格继续领导这一核心，并承担修改后的核心的额外责任。 虽然他作为LRI主席的角色非常耗时，但技术人员经验丰富，一直以来 与计划项目的核心相关至少两年。而核心人员将是 受训协助所有活动以满足需求，他们的主要活动如下：Angela Money隔离 人类血小板和内皮细胞；Alona Merkulova制备小鼠血小板和内皮细胞；以及 张晓伦在原核生物和真核细胞培养中表达蛋白质，并分离表达的蛋白质。 张先生在这些活动中有超过15年的经验。为方便起见，核心位于离子二 NB2上的房间，与迪科莱托博士的办公室和实验室以及秦博士的实验室同一层。其中一台配备了 组织培养罩和孵化器，第二个有蛋白质纯化设备。优先顺序将是 通常由迪科莱托医生设定。如果对核心活动的需求超过了供应能力，将讨论这一问题 在我们每月的计划项目会议上。任何未解决的优先级决定将由本计划做出 导演。

1. Title: Cell Isolation, Culture and Protein Expression Core 2. Core Unit Leader: Paul, DiCorleto, Ph.D., Chair, Lerner Research Institute, Cleveland Clinic Technical Staff: Xiaolun Zhang, Alona Merkulova and Angela Money. 3. Description The purpose of this core is to provide cells and proteins as needed to the four Projects of the Program. The specific cells to be isolated by Core B personnel are: 1) blood cells, primarily platelets of human and murine origin; 2) endothelial cells of human and murine origin; and 3) eukaryotic or prokaryotic cells expressing certain integrins, cytoskeletal proteins and mutants of these proteins. The prokaryocytic cells are used as a source for purified proteins; talin, kindlins and scr-family kinase derivatives, in the core and provide to the individual projects as needed. The preparation of cells by experience personnel will help to insure uniformity of purity and function. To have a reliable source of functional platelet preparations as needed by the projects is particularly valuable since the function of platelets can be highly variable. This is also true of endothelial cells of murine origin where experimental progress often depends on performing experiments with the cells derived from a small number of valuable mouse strains. The purified proteins are used in binding studies and for structural analyses, where relatively large quantities of high quality preparations are needed. With the need to prepare a limited number of proteins, isolation procedures can be standardized to provide high quality products. To perform these activities centrally will conserve resources, will help to insure quality and will allow for interproject comparison of results. In addition, since the individual Projects will still require substantial independent cell culture and will undoubtedly need to isolate cells for specific experiments, the Core will function in training members of the Program Project laboratories in proper cell culture and isolation methodologies and in appropriate ways to assess the quality of the cells. Thus, the Core serves an educational function as well. In the previous funding period, the Cell Culture and Expression were two separate entities. They are now consolidated into a single Core. This consolidation allows the new Core to provide more equitable service to all four Projects. Previously, Projects 2 made little use of the Cell Culture service and Projects 3 and 4 did not utilize the Protein Expression Core. Importantly, the consolidation also allows us to reduce the total annual direct costs from $250,426 for the prior Core B + C to $136,616 for the new Core B. This reduction is necessary because of annual budget rescissions and is feasible because the activities of the Core are conducted in adjacent space, allowing the core personnel to share responsibilities. Also, with the budgetary saving, we have been able to create a new In Vivo Core C ($111,900), which provides important services that will be used broadly in the Program. Dr. Paul DiCorleto serves as the Core Director. Dr. DiCorleto directed the Cell Culture Core within our program, which previously provided peripheral blood cells and endothelial cells to the Program Project. He is well-qualified to continue to lead this core and assume the additional responsibilities of the modified Core. While his role as LRI Chair is very time consuming, the technical staff is highly experienced and have been associated with the Cores of the Program Project for at least two years. While the core personnel will be trained to assist in all activities to meet demand, their primary activities are as follows: Angela Money isolates human platelets and endothelial cells; Alona Merkulova prepares murine platelets and endothelial cells; and Xiaolun Zhang expresses proteins in prokaryote and eukaryote cultures and isolates the expressed proteins. Mr. Zhang has more than 15 years experience in these activities. For convenience, the Core is located ion two rooms on NB2, the same floor as Dr. DiCorleto's office and lab and Dr. Qin's lab. One of these is equipped with tissue culture hoods and incubators, and the second has equipment for protein purification. Prioritization will be set routinely by Dr. DiCorleto. If demand for core activities is exceeding ability to supply, this will be discussed at our monthly Program Project meetings. Any unresolved priority decisions will be made by the Program Director.