Brewer s thioglycollate

Mice were euthanized by CO₂, 4 days after injection of Brewer’s thioglycollate (Sigma-Aldrich). A quantity of 5 ml of 4 °C Dulbecco's phosphate-buffered saline (DPBS) were twice injected into the peritoneal cavity of ApoE−/− mice with or without ANGII-induced AAA. Macrophages were then withdrawn from the intraperitoneal cavity and put into suspension, spun down (1,500 r.p.m. for 5 min at 4 °C) and 5 ml of RBC lysis buffer was added to the pellet. After incubation and spinning, 1 ml of 37 °C RPMI 1640, 10% FBS and 1% penicillin/streptomycin were added per mouse to the pellet. After counting and dilution (10-fold), cells were plated, analysed and used for further experiments.

As described (Javid et al., 2016), peritoneally recruited neutrophils were obtained by peritoneal lavage 2–3 hr after intraperitoneal injection with 1 ml of 5 mM sodium periodate. Neutrophils were counted using a Z1 Coulter particle counter (Beckman Coulter, Fullerton, CA, USA). PMs were obtained by peritoneal lavage 24 hr after intraperitoneal injection of 1 ml of filter‐sterilized 1% brewer's thioglycollate (Sigma). Peritoneal lavage in euthanized mice cleaned with 70% ethanol was performed by intraperitoneal injection of 8–10 ml pre‐warmed 1X dPBS−/− without calcium chloride and magnesium chloride (Sigma), incubation for 1–2 min, and collection of peritoneal fluid using transfer pipettes via a ventral midline incision. If no color change occurred, a second lavage with 1–3 ml dPBS−/− was performed. Sample purity was measured by Shandon Kwik‐Diff stain (Fisher Scientific, Nepean, ON, Canada) according to manufacturer's instructions; purity in all samples was >90%. Macrophages were enumerated using a hemocytometer.