iNOS protein expression in stimulated NK cells. NK cells were stimulated for 24 hours in the presence or absence of IL-12 (5 U/mL) or/and TNF (20 ng/mL) and then harvested. The cells were washed, permeabilized, and stained with a polyclonal rabbit anti-iNOS and then with a (FITC)-conjugated F(ab’)₂ fragment goat antirabbit IgG, as described in Materials and Methods. Fluorescence data were collected on 5,000 viable cells. (Open area) untreated NK cells (profile identical to that obtained with the preimmune serum, indicative that these cells were negative for the expression of this marker). (Shaded area) NK cells stimulated with TNF (upper panel), IL-12 (middle panel), or both cytokines (lower panel). The percentage represents the change in the mean log fluorescence for this experiment. Student’s t-test after comparison of the values obtained with IL-12 + TNF with the corresponding values obtained with medium, IL-12, or TNF results in P < .05. Similar results were obtained in three other experiments.