Lps escherichia coli o111 b4

Balb/c mice (males) were used at ∼8 weeks of age and supplied by Janvier Laboratories (Le Genet Saint Isle, France). Mice were anesthetized by intra-peritoneal injection of a mixture of ketamine-xylazine and were placed supine. For lung infection experiments, each specified strains of P. aeruginosa was grown overnight in Luria-Bertani broth and further transferred into fresh medium and grown for 4–5 h to mid-log phase. The bacteria cultures were centrifuged at 4000 × g for 15 min and the cell pellets washed twice with PBS. The bacterial pellet was diluted in its original volume and the OD adjusted to give the desired inoculum. A 50-μl bacterial suspension (1.10⁸ cfu/mice) was administrated by intranasal instillation and mice were then immediately held upright to facilitate bacterial inhalation until normal breathing resumed. The inoculum was verified by serial 10-fold dilutions of the bacterial suspensions and plating on LB agar plates. For the LPS challenge experiments, a 50 μl PBS suspension containing 3 μg of Escherichia coli O111:B4 LPS (Sigma-Aldrich, L2034) was administrated by the intranasal route, concomitantly with 5 μg of either active or heat-inactivated (InacLoxA, 10 min 95°C) recombinant Pseudomonas aeruginosa 42A2 lipoxygenase (provided by X. Carpena). BALF were performed 24 h after instillation, after a pentobarbital euthanasia procedure.