Isolation and Characterization of Mouse Neutrophils

Wild-type C57BL/6, Rab27a^+/+, Rab27b^+/+, ashen Rab27a^ash/ash, Rab27b knockout (Rab27b^−/−) and Rab27a/b double knockout (Rab27DKO) mice were bred in-house and generated as described previously [21 (link),26 (link)]. All animal experiments were performed with the approval of an ethics committee and in compliance with the UK Home Office Regulations under PPL 70/7078 at the Central Biomedical Sciences of Imperial College, London, UK. Neutrophils from mouse bone marrow were purified as described previously [36 (link)]. In brief, bone marrow cells were flushed from femurs and tibias of mice with PBS and red blood cells lysed by resuspension in a solution of 0.168 M NH₄Cl, 10 mM KHCO₃ and 0.097 mM ethylenediaminetetraacetic acid (EDTA). Cells were washed once with phosphate buffered saline (PBS), resuspended in 1 mL of PBS and layered on top of a discontinuous Histopaque gradient containing 3 mL of Histopaque (Sigma-Aldrich) at 1.119 g/mL at the bottom and 3 mL of Histopaque at 1.077 g/mL on top. Cells were centrifuged for 45 min at 700 g in a swing bucket centrifuge without braking. Cells at the interface of the two layers were collected and washed twice with PBS. Typical preparations contained above 80% neutrophils as assessed by Ly6G^high staining by flow cytometry.