CELL SORTER TO SUPPORT BASIC RESEARCH

本提案的申请人在生理，代谢和细胞信号传导领域进行前沿的基础脂质研究。迄今为止的研究已经解决了脂肪酸、磷脂、机械应力和运动如何影响全身生理、营养代谢和细胞信号传导的问题。这些基本问题已经解决了使用整体，细胞和组织培养方法的组合。人体绝大多数组织都是由各种各样的细胞类型组成的。这使得很难确定在给定的组织床内哪些特定的细胞对干预有反应。检查基因和蛋白质变化的组织分析的基本假设是，所有分离出来的细胞都是相同的，然而，这是一种严重的过度简化，而且还存在许多其他类型的细胞。例如，这些细胞可能包括基质细胞和上皮细胞或浸润性免疫细胞。离心和密度依赖的方法是可用的，但缺乏特异性和敏感性，因此需要大量的组织（即汇集来自多个动物的器官）来获得相对不纯的制剂。同样，在细胞培养方法中，通常需要根据治疗后的表型特征对细胞进行分类，然后评估所需的实验终点。这些实验需要只能使用配备细胞分选器的流式细胞仪来完成，而申请人并不容易获得这些基础设施。Bio-Rad S3e细胞分选仪是一种满足研究人员需求且价格具有竞争力的仪器。这是一个入门级的主力流式细胞仪，也有分选能力。唯一的其他直接竞争对手是索尼SH800。然而，虽然在功能上具有可比性，但它的价格没有竞争力。高端流式细胞仪的功能远远超出了这些研究人员的日常需求，而且成本也高达100万美元。预计获得该仪器将加强对4个实验室25+高质量人员的直接培训（每年）。此外，预计在圭尔夫大学人类健康和营养科学系建立之后，将有更多的教员及其学员寻求使用这一工具。认识到这一文书的重要性，该部将提供财政支助，以支持预算需要。总的来说，分离纯化细胞群的能力将有助于阐明作用机制，并改善许多高素质人才的培训环境。

The applicants of this proposal conduct leading edge basic lipid research in fields of physiology, metabolism and cell signalling. Studies to date have addressed questions about how fatty acids, phospholipids, mechanical stress and exercise influence whole body physiology, nutrient metabolism and cell signalling. These fundamental questions have been addressed using a combination of whole body, cell and tissue culture approaches. The vast majority of tissues in the body are comprised of a wide range of cell types. This makes it difficult to determine which particular cells within a given tissue bed are responding to the intervention. Tissue analyses examining gene and protein changes make the basic assumption that all cells isolated are the same, however, this is a gross oversimplification and a multitude of other cell types are also present. For example, these may include stromal and epithelial cells or infiltrating immune cells. Centrifugation and density dependent methods are available but lack specificity and sensitivity, thus requiring large amounts of tissues (i.e. pooling organs from multiple animals) to obtain relatively impure preparations. Similarly, in cell culture approaches it is often desirable to sort cells based on phenotypical characteristics following treatment and then assess the desired experimental endpoints. These experimental needs can only be accomplished using a flow cytometer equipped with a cell sorter, infrastructure that is not readily available to the applicants. An instrument that meets the needs of the investigators and competitively priced is the Bio-Rad S3e cell sorter. This is an entry level workhorse flow cytometer which also has sorting capability. The only other direct competitor is the Sony SH800. However, while comparable in features, it is not competitively priced. Higher end flow cytometers have capabilities far exceeding the routine needs of these investigators and also costing upwards of 1 million. It is anticipated that acquiring this instrument will enhance the direct training of 25+ high quality personnel (per year) across 4 laboratories. Furthermore, it is anticipated that additional faculty and their trainees will seek the use of this instrument once established in the Department of Human Health and Nutritional Sciences at the University of Guelph. In recognition of the importance of this instrument, the Department will provide financial support to support budgetary needs. Overall, the ability to isolate purified populations of cells will enable the elucidation of mechanisms of action and enhance the training environment for many high quality personnel.