Kinetics of appearance of NADPH oxidase–positive blood neutrophils after gene therapy in p47^phox−/− mice. Mice undergoing gene therapy were bled at 1 month after transplant and biweekly thereafter. Gene expression in PMA-activated neutrophils was detected by flow cytometry using the fluorescent probe dihydrorhodamine-123 (DHR). Data shown are the percentage of NADPH oxidase–positive blood neutrophils in experimental mice adjusted for the percentage determined in WT mice. (A) Mean percent of PB neutrophils that are NADPH oxidase positive in the DHR assay for nine experimental p47^phox−/− mice beginning at 4 weeks after gene therapy. The vertical axis is the mean percentage ± SEM of DHR⁺ circulating neutrophils in experimental mice. The horizontal axis represents time in weeks. This figure is derived from the data set shown in Table 1. (B) DHR flow cytometric dot-plot analysis of PB NADPH oxidase–positive neutrophils from one of the experimental p47^phox−/− mice included in the averaged data presented in (A). The vertical axis is neutrophil side scatter and the horizontal axis is neutrophil DHR fluorescence as a measure of NADPH oxidase activity. In each dot plot, 10,000 events (cells) are indicated with DHR⁺ neutrophils in a cluster to the right of the vertical line. The top dot plot displays the neutrophil DHR fluorescence from a representative WT mouse and is followed by sequential dot plots of the gene corrected p47^phox−/− mouse beginning 1 week before gene therapy (baseline) and then followed at 4-week intervals after gene therapy out to 20 weeks.