Neutrophil isolation

Neutrophil isolation was repeated three times in three separate experiments. The 8-week-old WT and PTX3 KO mice (one WT mouse and one PTX3 KO mouse per each neutrophil isolation) were culled via cerebral dislocation. The femur and tibia were then dissected out and maintained in Roswell Park Memorial Institute (RPMI) medium (Thermo-Fisher Scientific, UK) at room temperature (RT). The bone marrow was then extracted from the femur and tibia via flushing with RPMI medium through a 25-gauge (G) needle. WT and PTX3 KO suspensions were then vigorously triturated and centrifuged at 1000 g, 4°C for 5 min. Subsequently, the supernatants were discarded, and 6 ml of 0.2 % saline was applied for 1 min and triturated (to lyse red blood cells), followed by 14 ml of 1.2 % saline (to restore osmolarity) and vigorous trituration. Suspensions were then passed through a 70-µM strainer and centrifuged at 2000 g, 4°C for 10 min. Supernatants were discarded and pellets were resuspended in 5 ml HBSS (Thermo-Fisher Scientific, UK) and triturated. Suspensions were then added to 62% isotonic percoll (Sigma-Aldrich, UK) and centrifuged at 1000 g, 4°C for 30 min. Excess liquid was then removed and pellets were resuspended in 10 ml HBSS and triturated. Finally, suspensions were centrifuged at 2000 g, 4°C for 5 min, supernatants discarded, and pellets resuspended in 5 ml RPMI.