CXCL4 contributes to the <i>P</i>. <i>aeruginosa</i>-killing capability of neutrophils.

(A, B) The CXCL4 levels in neutrophils and platelets was evaluated after P. aeruginosa infection in vitro. Wild-type bone marrow-isolated neutrophils and blood isolated-platelets were left untreated (NT) or exposed to P. aeruginosa strain 8821 at a MOI of 1:10 for 6 h. Supernatants were subjected to ELISA analysis for determining CXCL4 production (n = 3 ± SEM, **p p -/- mice, and then incubated with P. aeruginosa 8821 (opsonized with mouse serum) at 37°C for 30 min. The cells were prepared using Cytospin. The specimens were then stained with Diff-Quik and examined under a microscope. The number of bacteria engulfed by 100 randomly selected neutrophils was counted. The phagocytic activity was measured according to the phagocytosis index (n = 3 ± SEM, *p-/- mice and incubated with P. aeruginosa 8821 (opsonized with mouse serum) at 37°C for 1 h. Gentamycin was added to kill extracellular bacteria for 3 h. Then, the neutrophils were washed and lysed with PBS containing 0.1% Triton X-100. The samples were serially diluted and spread onto Luria broth (LB) agar plates. Colony numbers were determined after overnight incubation at 37°C (n = 3 ± SEM, **p-/- bone marrow isolated neutrophil or blood isolated platelet were left untreated (NT) or exposed to P. aeruginosa strain 8821 at a MOI of 1:10. Supernatants were collected at 6h and analyzed for NO production (n = 3 ± SEM, ***p P. aeruginosa. Mouse bone marrow-derived neutrophils and blood-derived platelets were isolated from WT or CXCL4-deficient mice. An in vitro phagocytosis assay and intracellular killing assay were performed as described above. (n = 3 ± SEM, *p<0.05).