Cell Isolation, Culture and Protein Expression Core

1.标题：细胞分离、培养和蛋白表达核心 2.核心部门负责人：Paul，DiCorleto，Ph.D.，主席，Lerner研究所，克利夫兰诊所 技术人员：张晓伦、Alona Merkulova和Angela Money。 3.描述 该核心的目的是提供该计划的四个项目所需的细胞和蛋白质。由核心B人员分离的特定细胞为：1）血细胞，主要是人和鼠来源的血小板; 2）人和鼠来源的内皮细胞; 3）表达某些整联蛋白、细胞骨架蛋白和这些蛋白突变体的真核或原核细胞。原核细胞用作纯化蛋白质的来源;核心中的talin，kindlin和kindlin家族激酶衍生物，并根据需要提供给各个项目。由有经验的人员制备细胞将有助于确保纯度和功能的一致性。由于血小板的功能可能高度可变，因此项目所需的功能性血小板制剂的可靠来源特别有价值。这也适用于小鼠的内皮细胞 实验进展通常取决于用来自少量有价值的小鼠品系的细胞进行实验。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制剂。由于需要制备有限数量的蛋白质，因此可以将分离程序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，并可对结果进行项目间比较。此外，由于各个项目仍需要大量的独立细胞培养，并且无疑需要为特定实验分离细胞，因此核心将在适当的细胞培养和分离方法以及评估细胞质量的适当方法方面对计划项目实验室的成员进行培训。因此，核心也具有教育功能。 在上一个资助期，细胞培养和表达是两个独立的实体。现在，它们被合并到一个核心中。这一合并使新的核心能够为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没有使用蛋白表达核心。重要的是，合并还使我们能够将年度直接成本总额从以前的核心B + C的250，426美元减少到新核心B的136，616美元。这一削减是必要的，因为年度预算取消，而且是可行的，因为核心的活动是在相邻的空间进行的，使核心人员能够分担责任。此外，由于节省了预算，我们已经能够创建一个新的体内核心C（111，900美元），它提供了将在该计划中广泛使用的重要服务。 博士Paul DiCorleto担任核心总监。DiCorleto博士指导了我们的细胞培养核心， 该项目之前为该项目提供外周血细胞和内皮细胞。他是 完全有资格继续领导这个核心，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， 与计划项目的核心相关至少两年。虽然核心人员将在 经过培训，以协助所有活动，以满足需求，他们的主要活动如下：安吉拉钱隔离 人血小板和内皮细胞; Alona Merkulova制备小鼠血小板和内皮细胞;和 Xiaolun Zhang在原核生物和真核生物培养物中表达蛋白质，并分离表达的蛋白质。 先生张先生于该等活动方面拥有逾15年经验。为方便起见，核心位于离子2 NB 2的房间，DiCorleto博士的办公室和实验室以及秦博士的实验室在同一层。其中一个配备了 组织培养罩和培养箱，第二个有蛋白质纯化设备。优先次序将是 由迪科莱托医生进行例行检查如果对核心活动的需求超过了供应能力，将讨论这一问题 在我们每月的项目会议上。任何未解决的优先级决定将由该计划作出 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.