Fig. 3

Increased cytolytic granule accumulation in expanded NK cells. a The morphology of expanded NK cells. Cytospin preparations of NK cells (day 0, resting NK cells) and cytokine-activated NK cells (day 21) were stained by Wright-Giemsa staining. Representative images from cultures at 7, 14, and 21 days are shown. Original magnification × 400. b Immunofluorescence staining for perforin and granzyme B for NK cells before (day 0, upper panel) and after culture (day 21, lower panel) in the presence of a cytokine combination (IL-15/IL-18/IL-27). DAPI was used to stain the nuclei (blue). The negative control using secondary antibody (anti-IgG) only demonstrated low nonspecific binding of the secondary antibody. The data shown are representative of three independent experiments. Bars represent 10 μm. Original magnification × 100. c Western blotting analysis for perforin and granzyme B. NK cells were stimulated with cytokines (IL-15, IL-15/IL-18, and IL-15/IL-18/IL-27) for 21 days. The graph represents the relative expression of each protein. Protein bands were quantitated by densitometric analysis. The ratio of the intensity of protein bands relative to that of β-actin was calculated. Experiments were repeated three times with similar results. *P < 0.05, compared with cells cultured in the presence of IL-15 alone