Human CGD neutrophils demonstrate defects in actin polymerization, degranulation, and NET formation: restoration by H₂O_2. (A) Confocal microscopy. NET formation was analyzed after short-term stimulation (total 35 min) of control and CGD human neutrophils with the indicated triggers. Colocalization of elastase (green) or MPO (green) with released DNA (PI, red) is indicated by the yellow color depicting NET formation. (B) Flow cytometry. Expression of CD63, CD66b, and CD35 was assessed after pretreatment and short-term stimulation (total 35 min) of control and CGD human neutrophils with the indicated triggers in the presence and absence of 50 µM H₂O₂. Representative histograms are presented. (C) Confocal microscopy. F-actin distribution and morphological changes were analyzed after short-term stimulation (total 35 min) of control and CGD human neutrophils with the indicated triggers in the presence and absence of 50 µM H₂O₂. Right: Quantification of F-actin was performed by automatic analysis of microscopy images by using Imaris software. (D) Confocal microscopy. DNA release was analyzed after short-term stimulation (total 35 min) of control and CGD human neutrophils with the indicated triggers in the presence and absence of 50 µM H₂O₂. Right: Quantification of DNA-releasing neutrophils in ten high power fields. Data are means ± SEM. ***, P < 0.001; **, P < 0.01. n = 3. Bars, 10 µm.