Figure 3

Nucleoporated neutrophils conserve a responsive NADPH oxidase. A, Neutrophils were resuspended in solution "T" and transfected using an EGFP expression vector and an electrical setting corresponding to nucleofector program T27 (black lines) or incubated with DNA without electrical pulse (grey lines). The cells were recovered in serum-free RPMI containing 0.1% gelatin. Neutrophils (2 × 10⁶) were left untreated (left panel) or stimulated with PMA (right panel). Luminol-dependent chemiluminescence was continuously recorded for 65 min. The results are representative of three different experiments. B, Neutrophils were nucleoporated with an expression vector coding for EGFP, recovered for 2 h in RPMI and stimulated with PMA in the presence of Nitroblue Tetrazolium for 30 min at 37°C. After stimulation, cells were washed with PBS and fixed. Nuclei were visualized using the DNA-labeling fluorescent compound 4',6-Diamidino-2-phenylindole (DAPI). The samples were then analyzed by confocal microscopy and by differential interference contrast (DIC) microscopy.