Peritoneal lavage supernatants were thawed on ice and centrifuged at 12000rpm for 5′ at 4°C. The Biorad protein quantification kit (Bio-Rad Laboratories, USA) was used to normalize protein content. Samples containing 50 µg of total protein were assayed. VEGF and IL-1β cytokine levels were measured by ELISA according to the manufacturer's instructions (R&D Systems, USA).
Peritoneal fluid samples were diluted using water and methanol. The mixture was acidified with 2N HCl to pH 3.5 and applied to a C18 cartridge (Sep-Pak Classic, Waters®, IT) which was preactivated with methanol and water. The columns were washed with water and hexane, eluted by adding methyl-formate (1∶1 methanol and formic acid) and evaporated under a nitrogen stream. The residue was reconstituted in EIA Buffer and PGE2 quantification was performed using a monoclonal EIA kit (Cayman Chemical company, USA). Briefly, standards and samples containing 250 μg total protein were incubated overnight at 4°C in a plate pre-coated with goat anti-mouse IgG, with PGE2 Tracer (PGE2-Acetylcholineesterase conjugate) and mouse anti-PGE2 monoclonal antibody. The plate was subsequently washed, Ellman's reagent (5,5′-dithio-bis-(2-nitrobenzoic acid) and acetylcholine added and the absorbance was read at 405 nm.
Peritoneal fluid samples were diluted using water and methanol. The mixture was acidified with 2N HCl to pH 3.5 and applied to a C18 cartridge (Sep-Pak Classic, Waters®, IT) which was preactivated with methanol and water. The columns were washed with water and hexane, eluted by adding methyl-formate (1∶1 methanol and formic acid) and evaporated under a nitrogen stream. The residue was reconstituted in EIA Buffer and PGE2 quantification was performed using a monoclonal EIA kit (Cayman Chemical company, USA). Briefly, standards and samples containing 250 μg total protein were incubated overnight at 4°C in a plate pre-coated with goat anti-mouse IgG, with PGE2 Tracer (PGE2-Acetylcholineesterase conjugate) and mouse anti-PGE2 monoclonal antibody. The plate was subsequently washed, Ellman's reagent (5,5′-dithio-bis-(2-nitrobenzoic acid) and acetylcholine added and the absorbance was read at 405 nm.