NK cells isolated from a GS2 patient are impaired in the ability to recruit Munc13-4 to polarized perforin granules in PMA/ionomycin-activated cells. Resting NK cells were isolated from a patient, a healthy heterozygous parent, and a healthy donor, then were incubated alone or stimulated with PMA and ionomycin for 20 minutes at 37°C, followed by fixation, permeabilization, and labeling with anti-Rab27a polyclonal antibody and anti-perforin mAb. Confocal images of single, representative resting (A) and PMA/ionomycin-stimulated (B) Rab27a-deficient cells are shown, with scale bars representing 5 μm. (C) Colocalization of perforin with Munc13-4 was calculated as Pearson correlation coefficient, r, using the JACOP plugin in ImageJ. Ten to 15 cells per condition were analyzed. Mean values are plotted; error bars represent SD.