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

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.

This application is for the purchase of a FACS Canto II flow cytometer (Becton Dickinson). The equipment will be used by members of all RVC research groups for projects in a diverse range of fields covering basic immunology, cardio-vascular and reproduction biology in human and animals. Some projects require simple 2 colour analysis of common cell types while others require sophisticated 8 colour phenotyping of rare cell populations e.g. Treg. The FACS Canto II is state-of-the-art, with three lasers, which enable the detection of up to 8 colours simultaneously. This allows detection of rare cells and allows more flexibility in fluorophore use. For example, chylomicron remnant-like particles (CRLPs) labelled with the fluorophore, DiI, can now be enumerated (see Botham project). The analysis software is powerful but easy to use. The superior optics and sample uptake system (that captures up to 10,000 events per second) will enhance the resolution and rate of detection of dimly stained and rare cells. In addition the automated microtitre plate loader allows faster sample loading and less need for operator presence. Thus this machine will significantly improve the efficiency of flow cytometer use. The bench top design means that it is straightforward to house in our existing flow cytometry area. Importantly, the proposed machine can also utilise multiplex cytometric bead platforms, enabling cell subset analysis, functional studies and soluble and cell surface quantification. A key feature is the ability to detect multiple analytes in low sample volumes e.g. multiple cytokines concurrently. This increases the data that can be acquired from rare samples and enables financial and time-savings to be made. In conclusion, purchase of this equipment will strengthen the position of RVC scientists as internationally competitive researchers and contributing substantially to integrative whole animal physiology research of practical relevance to biomedical and veterinary sciences.