NADPH oxidase activation promotes digestion of neutrophil MPO within efferosomes. WT and CGD PEMs were pulse-fed hANs for 30 minutes, uningested hANs removed, and degradation of MPO determined in compared 6 hours by DAB histochemistry. (A) WT and CGD PEMss ingested similar numbers of hANs (30 minutes). Six hours postingestion CGD PEMs had significantly higher numbers of MPO⁺ macrophages compared with WT. (B) Averaged data from 2 to 4 experiments are shown. To compare relative digestion of ingested hANs, we determined the relative intensity of MPO staining in WT and CGD PEMs. Images were acquired using a 100× oil lens. Intensity of MPO staining was scored as “+++” (solid black arrows/strongly positive), “++” (dotted black arrows/intermediate intensity), or “+” (arrowheads/weakly positive) for each MPO⁺ efferosome at 6 hours and the percentage of distribution shown for each genotype. Results from 1 of 3 independent experiments are shown as mean ± SD. For panels A-C, at least 200 PEMs were scored for each genotype, per time point, and statistical differences between groups calculated using 2-way ANOVA with Bonferroni posttest correction. *P < .05; **P < .01; ϕP < .001. (C) PEMs were incubated with hANs for 30 minutes and chased for 6 or 24 hours. Lysates at the end of each time point were analyzed by western blot for MPO along with β-actin (loading control). Representative data from 1 of 5 independent experiments are shown. (D) Relative MPO band intensities normalized to β-actin were determined by ImageJ from samples from 3 independent experiments. Statistical differences between each time point calculated using the Student t test. ϕP < .001.