Isolation and characterization of mouse neutrophils

C57BL/6 mice were obtained from Charles River UK. PSD-95^−/− mice were obtained from the Wellcome Trust Sanger Institute. Female and male mice aged 7–12 weeks were used, with each serving as their own control. Neutrophils were isolated from either bone marrow or peritoneal exudates. Bone marrow derived neutrophils, which have similar respiratory burst and phagocytic characteristics to blood neutrophils [22 (link)], were extracted from age and gender-matched mice after cervical dislocation to avoid the potential confounding effects of inhalational anaesthesia on NMDAR activity [10 (link)]. Non-viable cells were excluded by 7AAD staining. Neutrophils were identified by Ly6G⁺Ly6C⁻CD11b^hi staining. For peritoneal neutrophil harvest, zymosan (1 mg·g⁻¹; Sigma, UK) was injected intraperitoneally in C57B/6 wild-type mice of either gender (age 6–8 weeks) prior to lavage with 5 ml ice cold PBS 3 h later. Highly purified neutrophils were obtained by negative selection using a neutrophil isolation kit (MACS Miltenyi Biotec), achieving ≥98% purity.