Percoll media

Percoll media (GE Healthcare, Bio-Sciences AB) of different densities were prepared as previously described [17 (link)]. A BM cell suspension or whole blood (3 mL) was gently layered on top of a two-layer discontinuous Percoll gradient: 1.093 and 1.077 g/mL (72 % and 60 % Percoll) densities for BM; 1.068 and 1.058 g/mL (51 % and 42 % Percoll) densities for blood. The tubes were then centrifuged at (1000 × g, 20 min, room temperature; Multifuge 1R, Thermo Fisher, USA) to separate the rat BM into three fractions. The middle-level cell fraction, which contained PMNLs, was washed twice in 10 mL of PBS (300 × g, 10 min, room temperature) to remove the remaining Percoll, and erythrocytes contaminating the pellet were removed using an Optilyse C lysis solution (Beckman Coulter, France). The PMNLs, separated by Percoll, were confirmed as BM neutrophil cell lines at various stages of differentiation by flow cytometry and microscopy using oil lens [18 (link)]. We used only samples containing >90 % BM PMNLs (bands and segmented granulocytes) and circulating PMNLs with 95 % cell viability, as determined by trypan blue staining.

Bone marrow leukocytes were isolated from mouse femurs. After sacrificing the mouse, both femurs were dissected and bone marrow was flushed with 10 ml RPMI by using a Ø 0.04 mm syringe. Neutrophils were isolated from the bone marrow using a density gradient separation method. The bone marrow was layered over Percoll media (GE Healthcare) followed by centrifugation (500 g, 30 min, 4 °C) to separate neutrophils. Erythrocyte lysis was performed using 9 ml 0.16M NH₄Cl and 1 ml 0.17M Tris HCl pH 7.5. Cells were washed and resuspended in phosphate-buffered saline for trypan blue cell viability assessment.