Computational protocol: Nanoscale kinetic segregation of TCR and CD45 in engaged microvilli facilitates early T cell activation

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[…] For calcium-flux experiments, Jurkat E.61 T cells were loaded with Fluo-4AM (Molecular Probes) at 5 μM for 30 min in the presence of 2.5 mM probenecid. T cells were transferred to imaging medium (RPMI without phenol red, 10% FBS, 25 mM HEPES) and allowed to adhere to the coated coverslips. We quantified Fluo-4 responses by determining the average intensity of a region within each cell as a function of time using the ImageJ program (NIH). [...] Data acquired by single-molecule localization microscopy was analysed by the N-STORM module in NIS-Elements (Nikon) or a previously described algorithm (ThunderSTORM) for the identification of individual peaks and group them into functions that reflect the positions of single molecules. Next, peaks were grouped and assigned to individual molecules for rendering. Peak grouping used a distance threshold and a temporal gap to account for possible molecular blinking. For fixed cells experiments, a temporal gap of ~50 ms and a distance threshold of 20 nm were applied for each fluorophore separately in order to minimize possible over-counting of molecules. Drift compensation and channel registration were performed using dedicated algorithms in the ThunderSTORM software. For live cells experiments no drift compensation was applied and a distance threshold of 20 nm was taken (regarding time gaps, each image in a live experiment accumulates 2–2.5 s of acquisition time as will be describe next) Three dimensional PALM was conducted and analysed using the astigmatism method. Calibration was conduct using 100 nm Tetraspec fluorescent beads (Invitrogen). Three-dimensional decoding was performed using Nikon NSTORM software. Individual molecules are presented in PALM and dSTORM images with intensities that correspond to the probability density values of their fitted Gaussian with respect to the maximal probability density values detected in the field. […]

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