p47^phox accumulation is decreased around S aureus phagosomes in p40^phox-T154A neutrophils. A quantity of 5 × 10⁴ wt or p40^phox-T154A neutrophils were incubated without or with 1 × 10⁶ serum-opsonized S aureus for 7 minutes at 37°C. Samples were cytospun onto glass coverslips, fixed, and immunostained for p40^phox or p47^phox as described in “Methods.” Mounted samples were visualized on a Zeiss LSM 510 META point-scanning microscope using fluorescence and DIC optics, as described in the Methods. Shown are representative fluorescence and DIC images for conditions tested. The position of internalized bacteria is indicated by arrows. Phagosomal accumulation of phox components was quantified using LSM 510 Image browser software. The average phagosomal fluorescence intensity per cell was calculated for n ≥ 71 cells, in 2-3 experiments, and was expressed as a fold increase in fluorescence intensity over cytosolic fluorescence intensity. Data were combined and are presented as fold increase in fluorescence intensity around the phagosome (% of wt; mean ± SEM). Where indicated, difference between means of wt and T154A is statistically significant. *P = .013 as determined by 2-way ANOVA. p40^phox: n = 87 cells for wt; n = 71 cells for T154A (3 experiments). p47^phox: n = 74 cells for wt; n = 84 cells for T154A (2 experiments).