Efferocytosis leads to NADPH oxidase activation and oxidant production in mouse peritoneal macrophages. (A) WT and CGD PEMs plated on chamber slides were fed hANs at 1:5 (PEMs to hANs) in the presence of NBT for 30 minutes. Black arrows point to efferosomes. NBT oxidation by ROS leads to purple formazan deposits, observed in WT PEM efferosomes, whereas formazan-negative cytoplasmic inclusions can be observed in CGD PEMs. Images were acquired using 100× oil lens. (B) hANs were labeled with Cell Tracker Red (denoted with a white asterisk) from an X-CGD patient were fed to WT mouse PEMs for 20 minutes. gp91^phox recruitment (green ring; white arrow) and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining (blue) was assessed by confocal microscopy. Images were acquired in a confocal microscope using 100× oil lens. Images from left to right denote confocal images taken at different planes (top to bottom). (C) PEMs from WT and CGD mice were stimulated with hANs, serum-opsonized zymosan (SOZ), and ROS production monitored using lucigenin-elicited chemiluminescence. Response kinetics are shown in panel C as mean ± SD from 1 of 5 experiments. Total integrated relative light units per second (RLU) over 1 hour from duplicate wells of WT or CGD PEMs after stimulation with hANs, apoptotic Jurkat cells, or SOZ from 3 independent experiments are shown for (D) total ROS and (E) intracellular ROS. (F) Total and (G) intracellular ROS production in WT and p40^{phoxR58A/R58A} PEMs after addition of hANs. Statistical differences between groups were calculated using 2-way analysis of variance (ANOVA) with Bonferroni posttest correction. *P < .05; ϕP < .01.