A state-of-the-art flow cytometer for a multi-disciplinary integrative biology department

流式细胞仪是生命科学研究实验室的核心工具。该提案包括学院四个研究主题中的三个项目，所有这些项目目前都使用流式细胞术技术。它们的范围从免疫学、病毒学、心血管和炎症生物学到生殖和发育。我们预计，如果这项建议获得资助，这些设备也将用于收集和分析其他一些项目的数据。流式细胞仪使用激光器和一系列偏转镜来通过检测细胞中的物理差异和/或通过检测可以附着到细胞内部或表面上的分子的荧光标记来区分不同的细胞群和微粒。这项技术对于计数血液或疾病部位的特定细胞类型至关重要，例如，验证外源基因导入细胞的成功。由于该技术的广泛应用，为了使我们的研究保持在国际科学前沿，我们需要日常使用的流式细胞仪，既易于使用，又处于技术的前沿。流式细胞仪已在RVC使用多年，并已被证明是非常宝贵的。然而，激光技术的最新进展以及新荧光染料和标记物的生产意味着我们目前的流式细胞仪Beckman Coulter EPICS XL-MCL无法执行许多当前研究项目所需的复杂分析。例如，我们目前的机器只能检测四种颜色，每秒分析1000个事件。此外，该学院从事免疫学研究的学术人员的显著增加意味着对这项技术的需求急剧增加。我们建议购买的机器是Becton Dickinson的FACS Canto II。与我们的EPICS相比，FACS Canto II可以检测八种不同的颜色，并以每秒10，000次的速度分析事件。与我们目前的机器相比，所提出的流式细胞仪的灵敏度也大大增加，并且能够测量和分析取自人类和动物的样品中的稀有细胞群。例如，嗜酸性粒细胞和T调节细胞（分别占总白色血细胞的1- 4%和3-8%），它们分别是Drs Lawrence和Garden实验室当前研究的主题，可以用该仪器容易地检测。它还将使广泛的用户能够进行复杂的研究，包括对细胞生长和细胞死亡的详细和量化分析，以及对感染和慢性疾病期间细胞产生的可溶性和细胞相关分子的分析。这台机器的另一个主要优点是，它将允许更有效地利用研究工作者的时间，因为不仅检测速度比EPICS快十倍，而且FACS Canto II的事件采集可以自动化。更强大的软件意味着对收集到的数据的分析将更加高效和准确。RVC特别强调学生获得实验室经验，因此共同申请人目前每年至少接待30名来自我们的BSc，BVetMed（插入或选修项目）和MSc课程的学生，并在任何时候监督大约20名博士生。此外，作为其四年博士培训方案的一部分，研究中心最近制定了一项由博士后科学家主持的特定实验室技术研究生培训方案。流式细胞技术的应用和实践经验将被纳入这一方案。因此，大量的学生将接触到这一尖端技术，同时进行实际的实验室为基础的项目作为他们的学位的一部分。

A Flow Cytometer is a central tool for life science research laboratories. This proposal encompasses projects from three of the four College research themes, all of which currently make use of flow cytometric techniques. They range from the fields of immunology, virology and cardio-vascular and inflammation biology to reproduction and development. We anticipate that should this proposal be funded, the equipment will also be used to collect and analyse data for a number of other projects. A flow cytometer uses a laser and a series of deflecting mirrors to discriminate between different populations of cells and microparticles either by detecting physical differences in the cells and/or by detecting fluorescent markers that can attach to molecules inside or on the surface of the cell. This technique is vital for enumerating specific cell types either in the blood or at the site of disease or, for example, to validate the success of introduction of foreign genes into cells. Due to the wide range of applications of this technique in order for our research to remain at the international forefront of science we require day-to-day use of a flow cytometer that is both easy-to-use and is at the cutting-edge of technology. A flow cytometer has been in use for many years at the RVC and has proved invaluable. However, recent advances in laser technology and the production of new fluorescent dyes and markers mean that our current flow cytometer, a Beckman Coulter EPICS XL-MCL, cannot perform the sophisticated analysis that many current research projects require. For example, our current machine can only detect four colours and analyses 1000 events per second. In addition, a significant expansion in academic staff at the College who undertake immunological research has meant the demand for this technology has dramatically increased. The machine we propose to purchase is the FACS Canto II from Becton Dickinson. In contrast to our EPICS, the FACS Canto II can detect eight separate colours and analyse events at a rate of 10,000 per second. The sensitivity of the proposed flow cytometer is also greatly increased compared with our current machine and enables the measurement and analysis of rare populations of cells in samples taken from human and animals. For example, both eosinophils and T regulatory cells (1-4 % and 3-8% of total white blood cells respectively) which are the subject of current research in the laboratories of Drs Lawrence and Garden respectively, can easily be detected with this instrument. It will also enable a wide range of users to undertake sophisticated studies including detailed and quantifiable analysis of cell growth and cell death and analysis of soluble and cell associated molecules that are produced by cells during infection and chronic diseases in a single small sample volume. Another major advantage of this machine is that it will allow more efficient use of research workers time as not only is the rate of detection ten times faster than the EPICS but acquisition of events by the FACS Canto II can be automated. The more powerful software means that the analysis of data collected will be more efficient and accurate. The RVC places particular emphasis on students gaining laboratory experience and therefore the co-applicants currently host at least 30 students per annum from our BSc, BVetMed (intercalating or elective projects) and MSc courses, as well as supervising approximately 20 PhD students between them at any one time. In addition the RVC, as part of its four year doctoral training programme, has recently instituted a programme of postgraduate training in specific laboratory techniques which is hosted by postdoctoral scientists. The application and practical experience of flow cytometric techniques will be incorporated into this programme. Thus a large number of students will be exposed to this cutting-edge technology whilst undertaking practical laboratory based projects as part of their degree.