Computational protocol: Actin polymerization‐dependent activation of Cas‐L promotes immunological synapse stability

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[…] TIRF imaging was carried out using a Nikon Eclipse Ti with × 60 NA 1.49 objective and an Andor DU897 back illuminated electron multiplying charge coupled device camera. Solid-state lasers (Coherent, Santa Clara, CA, USA) provided illumination at 488, 561 and 641 nm and narrow pass filters (Chroma Technology, Bellows Falls, VT, USA) were used for detection. Widefield epi-illumination for Ca2+ imaging was provided by a 150 W Xe lamp on an Olympus IX70 with a Zeiss × 40 1.3 NA Fluar objective, Ludl filter wheels and a Hamamatsu 12-bit C9100 1.1B charge-coupled device. Acquisition settings were maintained constant throughout each imaging procedure and between samples. Image analysis was performed with Metamorph and ImageJ. In brief, to measure intensities, images were subtracted for background, then cells in the subtracted images were marked with region of interests, and the intensity values obtained from the region of interests were plotted as raw values in scatter plots, unless otherwise indicated. The graphs and statistical analyses were performed with Microsoft Excel and Graphpad Prism, and P-values were calculated using two-tailed unpaired Student's t-test if the data were normally distributed or Mann–Whitney if data were not normally distributed. Data sets were tested for normal distribution using the Shapiro–Wilk test. Two-tailed Student's t-test was used for comparison between two groups. Microcluster tracking was performed with Volocity 4.2 (Improvision, Coventry, UK). [...] The cell boundaries were defined in ImageJ using the 'Magic wand' tool after making the images binary with the 'Threshold' function. The 'Magic wand' tool uses an algorithm that selects all connected pixels above the lower and below the upper threshold values (or value 1 in the case of a binary image), which were set based on the image background and pixel saturation, respectively. To obtain a coherent mask of the cells, the functions 'Close', 'Fill Holes' and 'Remove Outliers' were also used when necessary. Each cell was added to a list using 'ROI Manager', and then measurements of area and mean intensity were compiled. Our method to define cell boundaries involved using the program CellProfiler, setting a threshold intensity mask in one of the channels to identify the whole synapse area, and setting another mask on the other channel to identify TCR microclusters. Thus, we were able to measure total or mean fluorescence intensity of proteins over the whole synapse area, or just over the TCR clusters only. Using the masks defined in CellProfiler we computed the Pearson's correlation coefficient to quantify the overlap between phospho-Cas-L and TCR over the whole synapse area or just over the TCR clusters only. The Pearson's coefficient was then plotted to compare signal colocalization. […]

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