*2.2. Isolation of Mouse Peritoneal Neutrophils*

Induction of an enriched exudate of leukocytes in the peritoneal cavity of mice was performed by i.p. injection of 1 mL of aged, sterile 3% thioglycollate solution [16]. After 16 h, mice were euthanized, and the peritoneal cavity was flushed with 5 mL sterile Hanks' balanced salt solution containing 1% BSA (HBSS). The leukocyte pellet containing ~80% neutrophils and ~20% macrophages was washed with HBSS and resuspended in RPMI-1640 medium. Total cell counts were determined with a hemacytometer. Leukocyte viability was assessed by trypan blue exclusion (>99%). PMNs were then purified by adherence to a plastic dish as described by Tsurubuchi *et al*. [22]. Briefly, cells from peritoneal exudate were plated into a 35-mm plastic dish and incubated at 37 °C in 5% CO2 in air for 10 min in HBSS. The cells were washed twice with fresh HBSS to remove non-adherent cells. Although there was loss of some PMNs, which did not adhere to the dish, this procedure eliminated most of the macrophages. Cytochemical staining of adherent cells using HARLECO® Hemacolor® Solution (EMD Millipore, EMD Millipore) revealed that >95% of the adherent cells were PMNs.
