Engagement of LFA-1 preferentially induces Rab27a colocalization with perforin in resting NK cells, whereas engagement of CD16 preferentially induces Munc13-4 colocalization with perforin in resting NK cells. Resting NK cells were incubated with recombinant human ICAM-1-Fc or human IgG-coated beads for 20 minutes at 37°C, followed by fixation, permeabilization, and labeling. Cells conjugated with ICAM-1-Fc–coated beads were labeled with anti-perforin mAb and (A) anti-Rab27a polyclonal antibodies or (B) anti-Munc13-4 polyclonal antibodies. Cells conjugated with IgG-coated beads were labeled with anti-perforin mAb and (C) anti-Rab27a polyclonal Abs or (D) anti-Munc13-4 polyclonal Abs. Confocal images show single cells in conjugate with a bead and are representative of 5 independent experiments. Scale bars represent 5 μM. Colocalization between perforin and (E) Munc13-4 and (F) Rab27a antibody labeling in NK cells isolated from one representative healthy donor was measured and compared between resting NK cells, PMA and ionomycin-treated cells, and conjugates with IgG or ICAM-1–coated beads. Colocalization of the 2 fluorescent signals was calculated as Pearson correlation coefficient, r, using the JACOP plugin in ImageJ. Ten to 15 cells per condition were analyzed, and mean r is graphed, with error bars representing SD. (G-H) Plots represent cumulative data from 10 to 15 cells from each of 5 donors. Plus symbols represent mean values, and error bars represent SD. Boxes represent the 25th, 50th, and 75th percentiles. Two-tailed paired Student t tests were performed to assess significance.