Molecular Analysis of the Diversity of Lymphoid Cell Types and Functions at Single-Cell Resolution: ProteinSimple Single-Cell Western System.

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Protein composition of a cell defines its functional properties. Quantitative measurements of cellular proteins are therefore the cornerstone of most research programs in life sciences. It is increasingly recognized that an accurate understanding of cellular function requires studies with primary cells from the tissues, and can not be accurately modeled with transformed cell-lines in culture or other in vitro systems. However, most primary cells are found in very low numbers in the available biological samples, and are heterogeneous in nature. This is especially true for the cells of adaptive immune system, B and T lymphocytes. Each of these cells acquires an antigen recognition receptor of a unique structure and specificity during its development, and undergoes further differentiation, polarization, and functional specification during its engagement in immune response. Precise receptor stimulation thresholds dictate cell-fate, affecting how a lymphocyte engages in immune response or enforcing cell-anergy and apoptosis. The traditional experimental methods, like Western blotting, analyze bulk cell populations and do not accurately capture the functional complexity of the cells of adaptive immune system. The molecular analysis of rare cell types at single cell resolution is therefore critical for accurate understanding of lymphocyte functions and the mechanisms of adaptive immune response and immune regulation.******This application brings together a team of outstanding immunologists from across McGill University, studying the molecular mechanisms that regulate B and T cell functions in immune response, and facing the common challenge of protein quantification in small and heterogeneous biological samples.The Milo Single-Cell Western System, requested on this application, is the only instrument of its kind on the market allowing protein quantification in small biological samples at single cell resolution, and is not currently available at McGill University and the Montreal area. The equipment is therefore urgently needed and has the potential to fundamentally advance the research programs of the applicants, providing novel insight into the molecular mechanisms of B and T lymphocyte development and adaptive immune response. Such insight can not be obtained with existing or alternative equipment and methodologies.******Importantly, the applicants have an excellent track-record in HQP training, and currently their combined research teams include over 30 trainees. Most of these trainees are working on projects that require protein quantification in small and heterogeneous cell samples, and will therefore directly and strongly benefit from the requested Milo Single-Cell Western System. The benefits to the HQPs will include the direct acquisition of new technical skills, enhanced research productivity, and increased impact of their work on the research field. ******Overall, the requested equipment offers major benefits to the academic field and HQP training.**