Facilities

The MCCIR Flow Cytometry Facility is a full-time managed resource for flow cytometry technology. Flow cytometry allows high-speed quantitative analysis of individual cells and particles, and can be used to determine a number of physiologically relevant readouts such as antigen expression, signalling pathways, cell cycle, apoptosis and cytokine profiling.

Utilising lasers and fluorescence, labelled cells in suspension are 'sorted' into populations. Populations of interest can then be analysed using gates, or regions, within the software. Gated regions can be statistically analysed to yield information relating to relative percentages, fluorescence intensity and absolute counts.

The facility houses four multicolour analysis systems (BD FACSCanto II, BD LSR II, BD LSR Fortessa SORP and BD FACS Verse), a mass cytometer (CyTOF-Helios) and an imaging cytometer (ImageStream^X).

Our systems enable multi-parameter analysis of up to 18 fluorescent or >40 mass signals in a single sample, thus enabling the researcher to fully characterise their samples and/or define and rare cell populations within their samples. Imaging cytometry allows researchers to perform high speed, multi-parameter microscopy analysis of their samples.

In addition to the analysers, the facility houses a BD Influx cell sorter. Cell sorting allows the researcher to isolate and purify populations of interest from their samples. These isolated cells can then be used for downstream analysis or cloning.

With the ability to analyse and sort tens of thousands of cells a second the BD Influx can isolate very rare cells such as stem cells or low abundance immune cells. The BD Influx can sort up to six separate populations simultaneously and aseptically sorted cells can be collected in tubes or as single cells into multiwell plates. It can also be utilised to enrich fluorescent protein transfection experiments.

These state-of-the-art instruments allow MCCIR researchers to carry out refined multiparameter analysis and sorting of cell populations for both fundamental and clinical research, helping us better understand, characterise and quantify the complexity of the inflammatory response

We have facilities for ex-vivo modelling of the human lung. This includes air-liquid interface models using precision cut tissue sections from healthy and diseased lungs.

Cultures are generated from specific locations of the lung, including tracheobronchial, bronchial and bronchiolar regions, ensuring accurate cellular architecture is in place.

We are developing this system to include pulsatile flow of immune cells across the vascular layer to replicate blood and sheer pressure, and ventilator-induced barometric stretch on the alveolar layer to replicate breathing, so relevant airway secretions and physiology are in place.

We also have a modified cardio-pulmonary bypass system in place, allowing us to study entire human lungs with a range of aetiologies (chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and pulmonary hypertension).

For the first time, our unique lung systems give scientists the ability to map inflammation through initiation to resolution in translatable and relevant models of health and disease. Detailed assessments of drug safety, toxicology, and induced infection can be studied, along with gene transfer/knockout, and comprehensive cellular analysis.

The MCCIR houses novel facilities for fluorescence microscopy, including two platforms for super-resolution microscopy.

Classical optical microscopy is fundamentally limited in the details that can be observed due to diffraction – the way that light bends around objects. Super-resolution imaging techniques subvert this limitation and give is the ability to image cell biology on the nano-scale.

Our continuous-wave gated STimulated Emission Depletion (STED) microscope enables confocal imaging with the power to resolve features to 40 nanometres, in living cells, by confining the focal point of fluorescence with a unique doughnut-shaped laser beam.

With even greater resolving power, our Ground State Depletion (GSD) microscope maps the location of individual molecules within a cell – one by one – to reconstruct an image to 20 nanometres resolution.

These instruments allow us to see structures and elucidate nano-scale relationships between cellular components better than ever before. Members of the laboratory are using these super-resolution imaging techniques to define new paradigms in immune cell research, with papers in Science Signaling and Nature Immunology:

• Science Signaling 2013 Jul 23;6(285):ra62. doi:10.1126/scisignal.2003947 PMID:23882121
• Nature Immunology 2011 Jun 5;12(7):655-62. doi:10.1038/ni.2049 PMID:21642986