Examples of the NK cell IS during cytotoxicity. (A) Coincubation and subsequent fixed-cell imaging of NK cells (NK-92) conjugated to a K562 erythroleukemic target cell using spinning disk confocal microscopy. The image shows the formation of a lytic synapse between the NK and target cells where polymerized actin (white) is relatively enriched at the lytic immune synapse. The lytic granules (green; labeled using perforin as a marker for lytic granule content) are converged around the MTOC (red), with some having polarized toward the target cell (cyan; indicated with a membrane dye) as shown by the MTOC near the immune synapse. Image was cropped to focus on a single conjugate. (B) Structured illumination–total internal reflection fluorescence (SI-TIRF) microscopy of the actin meshwork at the IS of an NK cell (YTS) that was plated and fixed on an activating glass surface and then subsequently stained for F-actin using phalloidin. The activating surface is composed of anti-CD18 for adhesion and anti-CD28 for activation, thereby creating a representative target cell on a glass surface, enabling an en face assessment of actin architecture during IS formation. (C) Time lapse SI-TIRF microscopy of a YTS cell on an activated surface. YTS cells were placed on an activating glass surface and fixed at certain time points (2, 5, 10, and 20 minutes) and stained for F-actin using phalloidin to examine the evolution of the IS. The cell initially touches down upon the glass surface and initially extends out lamellipodia and filopodia as it spreads. By 20 minutes, a mature IS is formed as shown by the intricate dense actin mesh. Scale bars, 10 μm.