Role of Akt2 in chemoattractant and particle-induced O₂⁻ generation. Neutrophils from WT, Akt1^−/−, and Akt2^−/− mice were stimulated with C5a (A) and O₂⁻ generation was recorded as counts per minute (CPS) on the basis of isoluminol-enhanced chemiluminescence. The traces shown are representative of 3 independent experiments. Because of the short duration of O₂⁻ production, peak values of chemiluminescence were quantified (B) and shown as percentage change (mean ± SEM) on the basis of 3 independent experiments. *P < .05, **P < .01 relative to WT neutrophils. (C) Uptake of fluorescein isothiocyanate (FITC)–labeled zymosan by bone marrow–derived neutrophils from WT, Akt1^−/−, and Akt2^−/− mice after a 45-minute incubation. Shown are images taken with bright field (top row) and fluorescent (bottom row, shown in black and white) microscopy. (D) The zymosan particles in 20 cells were counted and quantified, and no significant difference was seen among WT, Akt1^−/−, and Akt2^−/− neutrophils. Shown are mean ± SEM from 3 experiments. (E) Mouse neutrophils from WT, Akt1^−/−, and Akt2^−/− were stimulated with serum-opsonized, FITC-labeled zymosan (500 μg/mL), and O₂⁻ generation was recorded on the basis of luminol-enhanced chemiluminescence during the indicated time period. (F) WT neutrophils were pretreated with the Akt inhibitor X (SH X) at the indicated concentrations for 10 minutes and then stimulated with 500 μg/mL serum-opsonized, FITC-labeled zymosan. O₂⁻ generation was recorded during the time period as described previously. The traces shown in panels E and F are representative of 3 independent experiments. Quantifications of data in panels E and F are shown in panels G and H, respectively, as mean ± SEM, on the basis of 3 experiments.