Murine dorsal air pouch model

Dorsal air pouches were formed on male Balb/c mice (6 to 8 weeks old) by injecting subcutaneously 3 ml of sterile air in day 0 and day 3. Mice were then intraperitoneally injected with anti-ChemR23 mAb or hIgG1 control mAb at 1 mg/kg or with RvE1 (50 μg/kg) on successive days at d5 and d6. On day 6, inflammation was induced by intrapouch injection of recombinant murine TNF (50 ng) or carrageenan (1%/1 ml per pouch). Pouch lavages (PBS-EDTA 2 mM) were collected at 4, 8, 24, or 48 hours, and cells were stained for phenotyping by flow cytometry. F4/80 (BM8), Ly6G (1A8), and anti-mouse ChemR23 (clone 477806) were used to identify macrophages, PMN, and ChemR23 expression, respectively. PMN mortality was evaluated with a viability dye (Life Technologies). The extraction protocol and analysis of bioactive lipids were performed as previously described (88) and adapted according to the Ambiotis SAS (Toulouse, France) standard operating procedure. The liquid chromatography–tandem mass spectrometry experiment was performed on an ExionLC™ AD Ultra-High-Performance Liquid Chromatography system (Sciex) coupled to a QTRAP 6500+ MS (Sciex) and equipped with electrospray ionization source and performed in negative ion mode.