Mouse Neutrophil Isolation

Blood was collected from the retro-orbital plexus of anesthetized mice in 1 mL of ethylenediaminetetraacetic acid (EDTA) anticoagulated buffer supplemented with 1% endotoxin-free BSA in sterile PBS, and peripheral blood neutrophils were subsequently isolated. Bone marrow–derived neutrophils were obtained by flushing the mouse femur 3-4 times with phenol red-free RPMI 1640 medium supplemented with 10 mM HEPES. The bone marrow–cell suspension was strained using a 40 µm cell strainer, and cells were pelleted by 10 minutes of 500 x g centrifugation before finally being resuspended in PBS.

Subsequently, peripheral or bone marrow–derived neutrophils were isolated by Percoll gradient centrifugation, as described elsewhere (7). Neutrophils were then resuspended in phenol red-free RPMI 1640 medium supplemented with 10 mM HEPES, and cell purity was assessed by Wright-Giemsa stain. After the neutrophil count was determined, the required cell density was adjusted by adding HEPES supplemented RPMI 1640 medium.