Computational protocol: Mechanism and function of Vav1 localisation in TCR signalling

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[…] Formation of T cell-APC conjugates, imaging and analysis were performed as described earlier (). Cells were stained with antibodies to Vav1 (2502, Cell Signaling) and phosphotyrosine (4G10, Millipore). Polarisation of Vav1 or phosphotyrosine was calculated as a recruitment index (RI) being the ratio of mean fluorescence intensity (MFI) at the APC contact site versus the MFI of the rest of the cell. For scoring the recruitment of molecules toward the APC, positive conjugates were defined as those where the RI of the molecules in question was at least 1 standard deviation (SD) greater than the mean RI in T cells conjugated to APCs bearing no antigen. Imaging of live 5C.C7 CD4+ or P14 CD8+ T cells expressing EGFP-Vav1 and subsequent classification of the patterns of accumulation of EGFP-Vav1 was carried out using image processing as previously described (). 5C.C7 T cells were conjugated with CH27 B cell lymphoma APCs pulsed with 10 µM MCC agonist peptide. P14 T cells were conjugated with B cells from C57BL/6 mice that had been activated for 3 days with anti-CD40 (0.5 µg/ml) and pulsed with 10 µM gp33 agonist peptide.To image microcluster formation, activated T cells were stimulated for 3 min at 37°C on coverslips (MatTek Corporation; P35G-1.5-10-C) previously coated with anti-CD3ε (2C11 clone) and in some cases also with anti-CD28 (37.51 clone) antibodies (both at 10 µg/ml). Poly-L-lysine coated coverslips were used as a negative control. Following stimulation, cells were fixed with 3% paraformaldehyde for 20 min at room temperature, washed twice with PBS, and permeabilised with 0.3% Triton X-100 in PBS for 2 min. Binding of primary and secondary antibodies was performed in blocking solution [PBS containing 0.1% saponin (Sigma, S4521) and 0.25% gelatin (Sigma, G7765)]. EGFP-Vav1 was detected using anti-GFP (chicken IgY, A10262, Invitrogen) with goat anti-chicken IgY NL493 (NL018, R&D Systems) as a secondary antibody. SLP76 was detected using an anti-SLP76 (rabbit polyclonal antibody, sc-9062, Santa Cruz) with AF568 donkey anti-rabbit Ig (A10042, Invitrogen) as a secondary antibody. TIRF imaging was carried out as previously described (), using an Olympus UPLSAPO×100 1.40NA objective and a custom build objective based TIRF system (). Excitation light was provided by a 20 mW 488 nm laser (Point Source, Hamble UK), and a 50 mW 561 nm laser (GLC-050-561, CrystaLasers, Nevada, USA), and illumination was synchronised with image capture using Winfluor software (http://spider.science.strath.ac.uk ). Analysis was performed using ImageJ and JACoP software. Area fraction was calculated as area of all clusters/area of cell. […]

Pipeline specifications

Software tools WinFluor, ImageJ, JACoP
Application Microscopic phenotype analysis
Chemicals Calcium, Guanine Nucleotides, Receptor-CD3 Complex, Antigen, T-Cell