In vivo models of peritonitis

Zymosan-induced peritonitis was induced as described previously with modifications.⁴⁸ Briefly, a solution of 20 μg/mL zymosan A from Saccharomyces cerevisiae (Sigma, St. Louis, MO) in sterile phosphate-buffered saline (PBS- Corning, NY) was prepared and autoclaved at 121 °C for 20 min. Two-hundred microliters of zymosan suspension (5 μg of zymosan) was administered by intraperitoneal (i.p.) injection. Mice were divided in two groups, untreated (injected with PBS only) or 2 h post-injection with zymosan. Mice were euthanized by exposure to isoflurane and peritoneal exudate was harvested by lavage with 5 mL of sterile cold PBS supplemented with 2 mM EDTA (Lonza, Rockland, ME) followed by cervical dislocation. Peritoneal exudates were centrifuged at 300 × g for 10 min at 4 °C: pellet-containing cells was processed for flow cytometric analysis while the supernatant was collected for chemokine/cytokine production measurement by enzyme-linked immunosorbent assay (ELISA). For lipopolysaccharide (LPS)-induced peritonitis, 250 μL of LPS suspension (from Escherichia coli O111:B4; Sigma) at 10 ng/mL was administered by i.p. injection. Mice were treated for 2 h with LPS. For TNFα-induced peritonitis, 250 μL of animal-free recombinant murine TNF-α suspension at 10 ng/mL (PeproTech, Rocky Hill, NJ) was administered by i.p. injection. Mice were treated for 4 h with TNF-α.