Fig. 4

Automated cell tracking of NK cell trajectories using the 2D live-cell imaging method provides valuable information about cellular behavior during target cell elimination. (A) The movement of each NK cell in the field of view was tracked and classified into three different types: small, intermediate, and large. Each color assigns a single NK’s trajectory during 120 min of the time-lapse video, overlayed on the representative frame. Scale bar = 100 μm. (B) Definition of trajectory diameter (dotted yellow) and trajectory length (dashed purple) using an example of the displacement of one NK cell (blue) recorded over 120 min. (C) Upper panels display the NK trajectories in control and pTNF-treated samples in one field of view of a well where the coordinates of x- and y-axis are given in pixels [px]. Lower panels display the percentage of different NK movements in control (Φ) and pTNF-activated PAECs. (D) Correlation between individual NK cells’ trajectory length and diameter (both in µm) is shown for all detected trajectories (left), with a zoomed-in view of those with diameters smaller than 20 μm (right). Comparison in NK behavior in response to control (Φ) and pTNF-stimulated PAECs is illustrated using violin-plots for (E) the trajectory length-to-diameter ratio, (F) NK cell average speed, and (G) the NK arrest coefficient for each of the three trajectory diameters. Data were analyzed from the average of NK cells per field of view and three technical replicates, with the total of at least 1,500 NK cells per group. Paired t-test was used for statistical analysis.