Novel Imaging and Data Analysis Techniques for Quantifying and Reporting Immunological Synapses: Pairing Amnis® FlowSight® technology and IDEAS® Software with FCS

Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involves reorganization of the cytoskeleton and recruitment of adhesion and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse after T cell receptor stimulation are required for the antigen-specific response. One way to measure an immunological synapse is by fluorescently labeling the molecules that have been recruited to the synapse and imaging via confocal or conventional fluorescence microscopy. However, immunological synapses are often rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. In this study, the Amnis® FlowSight® imaging flow cytometry platform was employed to collect imagery of large numbers of T cells and antigen presenting cells (APC) incubated with varying doses of antigen to assess the percentage of T cells involved in an organized immunological synapse. FCS ExpressTM Image Cytometry software was used to report and graph donor variation and dose dependent results with real time gating and statistics for quick and robust analysis across all samples. Advanced data analysis techniques in FCS Express combined with imaging flow cytometry technology allows researchers to more rapidly assess the results of experiments with visual cues amongst a large sample size and with “real time” access to statistical results as populations and samples change. Materials and Methods: