A Multi-User Flow Cytometry Facility for the Biosciences in Leeds

The ability to analyse specific cells from a mixture is fundamental to numerous cutting edge experiments in biology, ranging from the characterization of rare cancer causing cells to the isolation phosphate-accumulating strains of algae for use in fertilisers. The technique of flow cytometry (FC) has revolutionized our ability to characterise individual cells. It involves labelling cells with dyes that emit light (the process called fluorescence) when irradiated with light of defined wavelength, for example by binding dye-labelled antibodies to cells. These labelled cells are then detected by sophisticated fluorescence-activated cell analysers, using lasers to determine not only the number of dye-labelled cells but also the intensity of the labelling. Often cells are labelled with a number of different dyes, each activated by different wavelengths of light, and thus it is possible to further sub-divide populations of cells based on the combinations of dyes present. Indeed specific populations of cells can be isolated by deflecting the differently labelled cells to "sort" them into distinct populations.

Since its development in the 1960's, the dyes and machines used for FC have become increasingly sensitive and sophisticated, allowing very rare cells to be detected and also allowing these rare cells to be isolated as single cells, under sterile conditions. Indeed, current cutting-edge experiments require the ability to detect a broad spectrum of dyes as well as the newly developed "bright" labels to detect rare cells. Such experiments are not currently possible in Leeds due to the age of the machines and their inbuilt lasers. Here, we request state-of-the-art machines to build an integrated FC facility to address a series of fundamental questions in cell biology, infectious diseases and biotechnology. It is anticipated that these machines will precipitate a step-change in the sophistication of the experiments that we can perform. Specifically, the cell analyser requested can detect 13 different dyes simultaneously, allowing detection of very rare or "dim" cells whilst the cell sorter not only has an additional laser that is critical for detection of a very common red dye, but can also reliably sort cells under sterile conditions, which is essential to carry out cutting edge gene editing techniques.

These instruments will give momentum to a plethora of experiments, encompassing research groups from three different Faculties of Leeds University. This research ranges from the analysis of basic studies on the biology of cells, including how cancer cells form and develop, how the precursors to blood cells (stem cells) develop, how cells in the brain degenerate and how cells in the heart fail in heart disease. The instruments will also aid understanding of infectious diseases and how they can be combatted. These latter studies investigate important viruses like foot-and-mouth-disease virus that devastated UK agriculture in 2001 and human parasite infections such as malaria. Also the development of new medicines from bacteria and the mechanisms of bacterial resistance to antibiotics will benefit. Finally, the instruments requested will be important to a series of economically valuable biotechnology experiments that range from screening algae for their ability to take up phosphate for use as new fertilisers to the development of nanomedicines for cancer treatments. In all, 31 Leeds academics have research projects that rely on this equipment. When their research fellows, students and collaborators are included, over 100 researchers and their associated research projects will directly benefit from this new equipment.

Flow cytometry (FC) is a key technique in fundamental and applied aspects of molecular and cell biology. FC permits rapid detection and quantification of cell-surface and intracellular molecules based on fluorescent antibody staining or fluorescent fusion proteins. In addition FC forms the basis of cell-based assays such as apoptosis using fluorescently-tagged annexin V binding. Cell sorting by FC provides rapid purification and single cell cloning of cells that can be selected on the basis of fluorescence.
We have successfully and sustainably operated an FC Facility in the University of Leeds for 12 years. We now aim to effect a step-change in the research that can be performed with a state-of-the-art cell analyser (the Cytoflex S) and a cell sorter (the FACSMelody) and request funding for their purchase. These instruments will, respectively, detect and sort cells that express any of a wide spectral range of fluorescent fusion proteins based on a red fluorescent protein (RFP or mCherry, which our current equipment cannot detect), or enhanced green fluorescent protein (EGFP) or combinations thereof, as well as a wide palette of chemical fluorophores. The equipment requested has the capacity to detect and sort single cells aseptically which will have great impact on groups performing state-of-the-art genome engineering with CRISPR/Cas as well as in infectious disease research where biological containment is important. The equipment will be able to analyse and sort plant, animal and bacterial cells, as well as nanoparticles of 200nm or less.
We describe a broad range of interdisciplinary projects that will be transformed by access to the new equipment in the areas of fundamental mechanisms in cell biology, infectious diseases and biotechnology and include 11 investigators and 21 colleagues from the Faculties of Biological Sciences, Medicine and Health, and Mathematics and Physical Sciences as well as other collaborators and industrial partners.

The immediate, short-term impact of this cutting edge equipment will be on research with other academics. In addition, three companies/innovation centres with established collaborations to the PI/Co-Is will directly benefit, in both the short term as well as in the medium to longer term:
a) Badrilla, an established life science company aims use the new flow cytometry facility to clone antibody genes reactive to cardiovascular targets
b) Blueberry Therapeutics who actively collaborate with a Co-I on a biotechnology project to deliver RNA aptamers as novel topical disease modifiers for inflammatory skin disease
c) The Centre for Process Innovation (CPI) supports the UK process manufacturing industry, collaborating with large corporates, SMEs and universities to overcome innovation challenges and develop next generation products and processes. CPI wishes to access the new flow cytometry facilities.
In the medium to long term, other non-academic beneficiaries fall into three main classes:
1) The wider public
2) The commercial private sector
3) Charities within the non-public sector

The main ways in which they are likely to benefit from the more sophisticated research that will be made possible by the new flow cytometry facility are:
1) Improving health and wellbeing. In the medium term, this will be achieved by the successful execution of projects such as those to develop nanomedicines and the development of natural therapeutics from bacteria. In the longer term, the huge range of projects aimed at better understanding of the fundamental mechanisms of cancer cells, neurodegenerative diseases, cardiovascular cells, the effects of diet on liver metabolic health, as well as the molecular pathogenesis of viral, bacterial and protozoan infections, are likely to lead to the development of improved therapeutics and preventative measures.
2) Improving research capacity in businesses and organisations. In the medium term, the commercialisation of nanomedicines or natural therapeutics from bacteria outlined above will directly increase research capacity in the Biotech industry. In the longer term, fundamental research projects described will generate knowledge that underpins a wide range of research projects in the pharmaceutical industry as well as that funded by charities, especially CRUK, Wellcome, BHF and Gates.
3) Wealth creation. In the medium term, the biotechnology projects in particular are likely to lead to wealth creation. For example, the screening of algae for phosphate hype-accumulating strains can be used to clean up waste streams and potentially recycle the nutrients to fertiliser. Likewise, the more rapid screening of Affimers that will result from use of this equipment will help to further establish use of this technology globally. In the longer term, successful development of therapeutics from any of the projects investigating cell biology or infectious disease will similarly lead to wealth creation.
4) Cultural enrichment. Since many of the research projects described address fundamental biological processes that are likely to be of general interest to the public, they will therefore lend themselves well to public engagement.
5) The general public and school students will benefit from this equipment through gaining a greater understanding of modern scientific methods and the research that can be undertaken using such techniques. The University has developed its Public Engagement Strategy and sponsors a number of established large scale public engagement events. The PI/CO-Is have experience of delivering such outreach events.