The extended survival of conditioned neutrophils in vitro allows their genetic manipulation. (A) pmaxGFP plasmid (2 μg) nucleofection (Y-01 program) was performed on freshly purified neutrophils conditioned for 20 hours prior to analysis. Cells were imaged by confocal microscopy (whole neutrophil population) detecting GFP (green), Annexin V (Red), and DNA (PI, blue). Bar represents 60 μm. (B) The transfection efficiency was measured by flow cytometry on the Annexin⁻/PI⁻ population (left) comparing conditioned or unconditioned neutrophils (21% O₂, no supplementation) (right). Error bars indicate SD. ***P < .001 (n = 3). (C) The neutrophil viability (Annexin V⁻/PI⁻) and absolute cell count were calculated on control (untransfected) and transfected neutrophils (unconditioned/conditioned). Error bars indicate SD. ***P < .001 (n = 3). (D) The efficiency of siRNA transfection on conditioned neutrophils was evaluated on the LPS-stimulated IL-8 expression model. IL-8 mRNA expression was quantified upon stimulation with 10 ng/mL LPS during 3 hours at 37°C on freshly purified and conditioned neutrophils by quantitative reverse transcription polymerase chain reaction. Similar LPS stimulation was performed on neutrophils nucleofected with IL-8 siRNA or negative control siRNA (1 μM). IL-8 mRNA expression fold change was calculated compared with freshly purified neutrophils. Error bars indicate SD. “ns” indicates P > .05; ***P < .001, (n = 3). (E) Enzyme-linked immunosorbent assay quantification of IL-8 release in the supernatant fractions (Sup.) of neutrophils populations described in panel D. As controls, p65 and actin were detected by western blot in cellular (Cell.) fractions. Error bars indicate SD. ***P < .001; “ns” indicates P > .05 (n = 3).