| Summary: | Every cell in the human body is unique. In the immune system this is essential so that a wide range of invading pathogens can be recognised. To achieve the heterogeneity in lymphocytes, immature T lymphocytes and B lymphocytes undergo ‘priming’ in the thymus and bone marrow to diversify the cell surface receptors. As such, bulk analysis of immune cells and overlooking outliers, such as cells exhibiting unusual characteristics or responses, could lead to interesting cell characteristics being disregarded. By tracking the activation profile of a T lymphocyte against an antigen presenting cell in a high-throughput manner at the single cell level, the variation in activation levels of individual T lymphocytes will be identified. This will lead to an improved understanding of why certain T lymphocytes are more efficient at eradicating diseases. This thesis describes the design and development of a bespoke microfluidic device able to trap thousands of individual cells in nanolitre wells, for the analysis of cell-cell interactions. The device is optically transparent, enabling T lymphocyte activation to be observed in real time using fluorescent microscopy and calcium staining; thousands of cells may be observed within a single microscope field of view. Cell-cell contact is controllable by the device, allowing the activation of T lymphocytes against antigen presenting cells to be viewed in real time, in a high-throughput manner. The device has been used to investigate T lymphocyte activation against soluble stimulants, as well as antigen presenting cells. Specifically, the temporal calcium response of single cells has been studied and experimental results are presented. The findings reveal differences in activation profiles of individual cells within a clone population, which are not evident using state-of-the art bulk cell analysis techniques such as flow cytometry. This work highlights the importance of assessing single cell responses in an immune interaction.
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