Instruments for checking physical characteristics

Description

The Francis Crick Institute (‘the Crick’) is a biomedical research institute dedicated to understanding the scientific mechanisms of living things. Its work is to understand why disease develops and to find new ways to treat, diagnose and prevent illnesses such as cancer, heart disease, stroke, infections and neurodegenerative diseases. The Francis Crick Institute requires two high-end 5 laser Flow Cytometers, (an analyser and cell sorter) that incorporate the very latest technologies available in the field, to enable the analysis and cell sorting of samples containing Dangerous Pathogens (ACDP) at containment 3 level. These samples include viable human and experimental infectious samples (e.g. cells infected with M. tuberculosis, HIV, influenza, malaria parasite or other). The instruments will be housed in a class 1 Biological Safety Cabinet. All of these experiments can frequently need up to 18 fluorochromes requiring excitation by a combination of 355nm, 405nm, 488nm, 561nm and 640nm lasers. Currently, the Crick is unable to conduct experiments on such live cells requiring handling at containment 3 and which are stained with up to 18 fluorochromes because we do not have suitable high end cytometers housed in a class 1 Biological Safety Cabinet with this detection capability. Therefore this is impairing our ability to maintain our cutting-edge international reputation and success in this research area at the highest international level. In research into the immune response to tuberculosis (Mycobacterium tuberculosis) and other pathogens such as influenza, HIV and malaria it is essential to be to be able to analyse, cell sort and subsequently identify both infected cells and those involved in the immune responses to these pathogens. An understanding of immune control of the pathogen and the factors resulting in disease pathogenesis is of fundamental importance for protection. Due to the complexity of the cells involved in immune regulation, both in infection and disease, populations of immune cells need to be analysed and/or cell sorted to determine their functional characteristics. A central part of this work is to gain a detailed understanding of the extent and kinetics of immune cell recruitment into the infected lung and /or other tissue, and the long-range effects of the pathogen in the host. The analysis of innate and adaptive immune cells requires staining cells with up to 18 fluorochromes. Flow cytometric analysis and cell sorting of the cell types recruited and of their activation state is crucial to do this type of experiment, and we are presently setting up 18 colour panels to analyse in detail adaptive and innate immune cell subtypes in situations where use of Containment Level 3 Flow Sorter is a requirement. Importantly, not all staining can be done on fixed cells as resolution is lost, therefore, certain antibody panels for multiparametric analysis will work only on unfixed cells. The ability to place them into a Cat 3 environment would allow us to perform this crucial part of our work enabling a better understanding of the immune cell types involved in pathogenesis and in protection in these important infection situations and tracing the pathogen which can be labelled with reporters where a fluorochrome is often fixation sensitive. In addition, specific B and T lymphocyte responses together with cells of the innate immune response are analysed directly ex-vivo throughout infections in many genetically manipulated mice for their multi- functional capabilities. All these experiments require the instruments to have the high sensitivity not available in other manufacturer's instruments and the capacity to analyse up to 20 parameters in order to detect rare cells. This can only be achieved using the new generation instruments. The Crick is particularly interested in multicolour detection of different cell types of the immune response which may be protective or on the other hand help to disseminate the pathogen throughout the body during infection and also for detection of mycobacterial fluorescent reporters at the single cell level as well as sorting and measurement of bacterial viability (e.g. by colony forming units) in both mouse and human cell samples. Therefore, to have access to this instrumentation on a regular basis in CL3 will is essential to maintain our international competitive edge in our research in tuberculosis tackling different aspects of pathogenesis and host adaptation. Crucially, we need instruments whose enhanced sensitivity is good enough to detect cells expressing fluorescent proteins. In the future, we will use a broad range of other fluorescent proteins such as mCherry and katushka, which will enable us to study co-factors that might be involved. The study of cytokine producing cells and their impact on a variety of immune responses are increasingly reliant on state of the art flow cytometry for accurate detection of these rare events. Additionally, we are expanding our studies to tissues with low cellular content where access to modern multicolour flow cytometry is a crucial prerequisite for such analysis. To track the development, differentiation and regulation of pathogenic cells in several diseases models, various mice with fluorescent proteins (GFP, YFP, RFP and Katushka) under the control of important gene promoters use used. Cells from these mice have to be analysed or cell sorted at containment 3. To keep up with the fast pace of research we critically depend on this technology, which is our main tool for investigating the function and characterisation of cells in in-vivo and ex-vivo biological systems requiring handling at containment 3 and is used in all our on- going research projects. There is a requirement for containment 3 flow cytometry from a variety of Labs in addition to those working on tuberculosis as described above—additionally those studying multiple phenotypes in heterogeneous samples from infected patients, those using virally-infected cells lines, those looking at rare events and functional studies. The Labs that would take advantage of this instrument will include those looking at tuberculosis infection and multiple other infections in humans. Many of these will deal with primary human cells, human cell lines or samples infected with tuberculosis or other pathogens. The ability to run samples without the need for fixation will allow better resolution of weakly expressed markers and allow functional assays such as calcium flux, other kinetic studies and apoptosis. In addition to containment, the groups will require access to cytometers that are capable of multicolour analysis and cell sorting of a wide range of fluorochromes and fluorescent proteins and need to be able to take advantage of the newer dyes that are becoming available. Particularly important is the ability to differentiate multiple fluorescent proteins in the same sample—currently CFP, GFP, YFP and RFP. Adaptability of any cytometer in containment is vital to cover the range of applications needed and to allow some future-proofing and expansion. For maximum efficiency the analyser and cell sorter must have the same configurations and software to allow for experiments to be run on either and therefore increase capacity. Without this high level instrumentation the Facility will be unable to meet the growing demand for containment 3 analysis and cell sorting on up to 18 colours, 5 laser instruments and thus maintain our international research reputation in this area.