Distribution of exogenously expressed p47^phox-PX domain and RhoB during phagocytosis in HL-60 human promyelocytic cells HL-60 promyelocytic cells were differentiated to granulocytes and transfected with the indicated expression vector as described under "Methods." Phagocytosis of Texas Red-labeled opsonized-zymosan particles was conducted at 37°C for 15 min. B, The difference between the fluorescence intensity at the phagosome membrane versus that at the surrounding cytosolic area (ΔF_I) was calculated using the NIH image processing and analysis program IMAGE/J software as described under "Methods." The results are mean ± SEM of three phagosomes from different cells.

EGFP-p47^phox translocates to the plasma membrane in nucleoporated neutrophils. Neutrophils were nucleoporated with a vector expressing EGFP-p47^phox or EGFP control vector and recovered in RPMI. Two hours after transfection the specimens were transferred to a live chamber (37°C and 4.9% CO₂) and cells were stimulated with PMA (0.1 μg/ml). The distribution of the fluorescent chimera was analyzed by confocal microscopy at the indicated intervals. The images were processed and analyzed for the distribution of the fluorescence intensity using the NIH image processing and analysis program IMAGE/J software. Scale bars = 3 μm.

The PX domain of p47^phox localizes at the plasma membrane in resting and stimulated neutrophils. Neutrophils were nucleoporated with the expression vector EGFP-p47-PX or with the EGFP control vector. After 2 h, the cells were stimulated with fMLP (1 μM), PMA (0.1 μg/ml) or left untreated. The cells were then fixed and analyzed for fluorescence distribution by confocal microscopy. The images were processed and analyzed for the distribution of the fluorescent proteins using the NIH image processing and analysis program IMAGE/J software. Scale bars = 3μm. For EGFP-p47-PX, separate cells are shown. For EGFP, the same cells are shown before and after stimulation. The results are representative of three independent experiments.

Transfected neutrophils are responsive to soluble stimuli. Neutrophils were transfected with the expression vector pEGFP-p47^phox or with the pEGFP control vector as follows: Neutrophils were resuspended in solution "T" in the presence of the expression vector pEGFP and DNA was transfected using nucleofector program T27. The neutrophils were transferred to a poly-L-lysine – coated chambered glass slide containing 10% FCS-RPMI. Two hours after transfection, the cells were stimulated with PMA (0.1 μg/ml) or fMLP (1 μM) at 37°C for 15 min or 5 min, respectively. The cells were fixed with 3.7% paraformaldehyde and analyzed by confocal microscopy. Transfected neutrophils stimulated with PMA or fMLP can undergo morphological changes represented by the apparition of protrusions of the plasma membrane (arrows). Scale bar = 5 μm. These results are representative of four separate experiments.

Optimization of transfection and gene expression in human neutrophils. A, Optimization of transfection and expression of the β-galactosidase gene in human neutrophils. Neutrophils were resuspended in the indicated amaxa solution, in the presence of the pCMV-β-gal expression vector (1–9) or the empty vector pCMV-PA (10), and transfected as described in "Methods." The programs used were as follows: 1, "A23"; 2, "A27"; 3, "T20"; 4, "T27"; 5, "T16"; 6, "T01"; 7, "G16"; 8, "O17"; 9, no pulse and 10, "T16". Transfection of pCMV-PA resulted in no detectable activity for any of the programs listed. These data represent the mean and SE of six separate experiments. B, Neutrophils (2 × 10⁶ cells) were resuspended in solution "T" and transfected with the expression vector pmaxGFP (amaxa biosystems) as described in "Methods" using the electrical settings corresponding to programs T27, U14 or Y01. After a 2-hour recovery period, dead cells were stained with propidium iodide and the samples were analyzed by flow cytometry.

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.

Surface expression of CD11b in nucleoporated neutrophils. The expression of the human leukocyte integrin subunit CD11b on the surface of nucleoporated neutrophils was analyzed by flow cytometry as described in "Methods." A, Cells were transfected using solution "T" and an electrical current corresponding to program T27. Nucleoporated cells were recovered for 2 h in serum-free RPMI medium (blue lines). Untransfected cells (no pulse) were recovered in RPMI and, where indicated, stimulated with fMLP for 5 min (red lines) or left untreated (green lines). The cells were labeled with anti-CD11b or an isotype-matched control. Expression of CD11b antigen on the surface of treated and untreated neutrophils was analyzed by flow cytometry. The results are representative of three independent experiments. B, Cells were transfected using solution "T" and an electrical current corresponding to program Y01 (blue lines) or not pulsed (green line). The cells were labeled for surface expression of CD11b as described above. The results are representative of three independent experiments. C, Cells were transfected with the expression vector pmaxGFP using the electrical setting corresponding to program Y01. After a 2 h recovery period, the cells were analyzed for the detection of the surface expression of CD11b using a specific antibody conjugated to phycoerythrin (PE, BD Biosciences). The samples were analyzed by flow cytometry as described under "Methods."

Distribution of exogenously expressed p47^phox-PX domain and RhoB during phagocytosis in human neutrophils. Neutrophils were nucleoporated with the expression vectors EGFP-p47-PX or EGFP-RhoB as described in figure 2. After a 2 h recovery period, cells were incubated in the presence of Texas Red-labeled opsonized-zymosan particles at 37°C for 10 or 15 min. Samples were washed, fixed and analyzed by confocal microscopy as described in "Methods." The images were processed and analyzed for the distribution of the fluorescent proteins using the NIH image processing and analysis program IMAGE/J software. The plot profiles, in the right panel, show that EGFP-RhoB is accumulated around the phagosome (red arrows indicating peak of fluorescence intensity) while the plot profiles corresponding to EGFP-p47-PX show a plateau of fluorescence intensity (red lines). The white arrows show the distribution of p47^phox-PX domain in membrane protrusions surrounding the opsonized particle at time zero. Scale bar = 3 μm. B, The difference between the fluorescence intensity at the phagosome membrane versus that at the surrounding cytosolic area (ΔF_I) was calculated using the NIH image processing and analysis program IMAGE/J software as described under "Methods." The results are mean ± SEM of three phagosomes from different cells.