Endogenous and heterologously expressed p67^phox or iPX are targeted to pre-phagosomal structures during phagocytosis of IgG-coated particles. (A) Primed WT neutrophils (5 × 10⁴) were incubated without or with 1 × 10⁶ serum-opsonized S aureus for 7 minutes at 37°C (i), or with 5 × 10⁴ unopsonized or IgG-opsonized beads for 20 minutes at 37°C (ii), as described in “Phagocytosis assays.” Samples were cytospun (i) or allowed to adhere (ii) onto glass slides, fixed, and stained for p67^phox as described in “Phagocytosis assays.” Mounted samples were visualized on a Zeiss LSM 510 META point-scanning microscope using fluorescence and DIC optics. Shown are representative fluorescence and DIC images for conditions tested. Phagosomal (i) and extra-phagosomal (ii; indicated by arrows) accumulation of p67^phox was quantified for at least 50 events under each condition using LSM 510 Image browser software and expressed as mean ± SEM as a percentage of cytosolic levels, *P < .0001, Student t test. (B-C) BMNs expressing p67^phox-GFP (Bi,Ci,Ciii) or GFP-iPX (Bii,Cii) in a WT genetic background were primed at a concentration of 2 × 10⁷/mL, placed on ice and gently mixed at regular intervals. Aliquots of 5 × 10⁵-1 × 10⁶ cells were settled onto glass coverslips in imaging chambers that were previously blocked with 100% FBS. Cells were incubated with serum-opsonized S aureus (1-5:1 S.aureus:neutrophil; B), or IgG-SRBCs (20:1 IgG-SRBCs:neutrophils; C). z-stacks of neutrophils undergoing phagocytosis were captured over time on a Nikon-Eclipse spinning disk confocal microscope equipped with an ultrasensitive EM-CCD camera, as described in “Live imaging.” A total of 20-22 slices were captured for each time frame, at a distance of 0.5 μm between each slice, encompassing the whole neutrophil. The exposure time was 100 milliseconds, and the time interval between each frame was 5.896 seconds (p67^phox-GFP), 10.52 seconds (GFP-iPX; Bii), or 11.046 seconds (GFP-iPX; Cii). 3D reconstruction was performed using Volocity software, and reconstructions at individual time frames representing different stages of phagocytosis are shown. The time of attachment of particle to neutrophil and/or the time of formation of the phagocytic cup was defined as t = 0. Stars indicated the position of particles during various stages of phagocytosis; white arrows indicate the position of bacterial phagosomes containing p67^phox-GFP or GFP-iPX (B) or pre-phagosomal accumulation of p67^phox-GFP or GFP-iPX (C). Imaging experiments were performed with neutrophils from individual mice, on at least 2 different days. (Ciii) Series of confocal images over time, showing fusion of a pre-phagosomal structure containing p67^phox-GFP with an IgG-SRBC phagosome.