161533 TriKE induces robust NK cell killing dynamics. Purified NK cells by negative depletion were stained with 0.5 μM CellTrace calcein red-orange AM and HL60 target cells with distinct dyes for 10 to 20 minutes. The blue-stained NK cells and green-stained target cells were seeded onto a silicon-glass microchip divided into 2 separate compartments containing 161533 TriKE or 1633 BiKE. Imaging of the microchip was performed using a Zeiss LSM 880 microscope equipped with an environmental chamber kept at 37°C and 5% CO₂. Images were acquired using a 10× objective every 3 minutes for 9 hours. (A-D) NK cell cytotoxicity (A), representative images of target cell killing (red cells) at different time points (B), time to first kill and migration (C), and serial killing (D) are shown from 5 independent experiments (n = 1031 NK cells for BiKE and n = 1202 NK cells for TriKE). Data were quantified by MATLAB, and statistical analyses were performed by paired Student t test (A-B) or Mann-Whitney U test (D).