NADPH oxidase deficiency is associated with delayed maturation of efferosomes. hANs were incubated with WT or CGD PEMs for 7, 15, or 30 minutes as indicated, and efferosome maturation assessed using confocal microscopy–based assays. Images were acquired in a confocal microscope using a 40× oil lens. Representative confocal images shown were taken at 7 minutes, with marker-positive efferosomes indicated by arrows and marker-negative indicated by arrowheads. The percentage of efferosomes positive for the specified markers were analyzed for each genotype (WT = blue; CGD = red) and shown in the panels to the right of each confocal image. (A-B) LC3 acquisition or (C-D) Lamp1 acquisition at 7.5 and 15 minutes. (E-G) Lysotracker Green was added to PEMs for 5 minutes prior to the indicated time points to compare relative acidification of efferosomes. Panel F shows relative fraction of Lysotracker-positive efferosomes in WT and CGD PEMs. (G) MFI in individual Lysotracker-positive WT and CGD efferosomes PEMs 15 and 30 minutes post hANs feeding in 1 representative experiment. MFI was determined using ImageJ. (H-I) To compare proteolysis rates, hANs were coated with DQ-BSA and subsequently fed to PEMs. (I) The fraction of brightly green fluorescent efferosomes in WT and CGD at 7.5, 15, and 30 minutes postfeeding, indicating cleavage of DQ-BSA and unquenching of the BOPIDY dye, whose fluorescence is insensitive to pH over a wide range. Cumulatively, 20 to 50 efferosomes in each of >3 experiments were analyzed for each time point studied for each treatment condition for WT and CGD. Statistical differences between genotypes were measured for each time point using the Student t test (mean ± SD). *P < .05; **P < .01; ***P < .001.