NK cell surveillance involves the dynamic phases migration, conjugation, lytic hit, and attachment. (A) Schematic representation of the process of NK-target cell interaction. Top: NK cell (red) migrating to form a conjugate with a target cell (green) leading to NK-mediated killing of the target cell as detected by decreased intracellular calcein fluorescence in conjunction with visible signs of death (blebbing or swelling). Middle: Intensity profile (blue line) of target cell intracellular calcein showing a clear decrease and decay after the lytic hit. Bottom: Time line defining the phases migration, conjugation, delivery of the lytic hit, and attachment, and the times that were assessed from the experimental data. Time (1) = time to lytic hit, time (2) = time to death, time (3) = conjugation time, and time (4) = attachment time). (B) Time-lapse imaging data showing fluorescence (top), bright-field (middle), and overlay (bottom). An NK cell (red cell body marked by white arrowhead) migrated (Mig, frame 1) to form a conjugate with a target cell (green, star; Conj, frames 2-5), followed by attachment (Att, frame 6) and resumed free migration (Mig, frame 7). At frame 3, the NK cells delivered a lytic hit triggering calcein leakage, which is seen as decreased green fluorescence intensity (frames 3-7) and visible signs of target cell death (frames 4-7). White lines in the overlay images show the NK cell trajectory. Indicated times are hours: minutes. Scale bar indicates 15 μm.