Figure 2. MyoIIA deficiency in activated T cells causes defects in trans-endothelial migration (TEM) under flow.

Control and MyoIIA KO activated T cells were fluorescently labeled, mixed at a 1:1 ratio and perfused into a flow chamber containing a monolayer of bEnd.3 brain endothelial cells and then kept under a physiological shear flow rate of 2 dyne/cm² for 30 min. During this time, phase contrast, green and red fluorescence images were acquired every 15 sec. A) Schematic showing the TEM flow chamber setup. B) Percentage of transmigrating T cells, calculated by quantifying the number of T cells that lost their phase contrast ring and underwent a stepwise darkening in the phase contrast channel during the time-lapse, relative to the total number of T cells adhered to the endothelial cell monolayer. Data are the mean (±SEM) of 3 independent experiments. C) Selected time-point images of representative control and a MyoIIA KO T cells during TEM. Phase contrast images (left) and fluorescence overlay on the phase contrast (right) are shown. White arrows point to T cells above the endothelial monolayer; black arrows point to T cells that have completed TEM and are under the endothelial monolayer. Time in min: sec.