Isolation of Neutrophils From Bone Marrow

Percoll-based (GE Healthcare, 17144003, Little Chalfont, United Kingdom) density gradient centrifugation was used for the purification of neutrophils from bone marrow cells (Swamydas et al., 2015). A “100% Percoll” solution is generated by adding 5 ml of 10X HBSS (Gibco, 14065056, United States) to 45 ml of Percoll. Percoll dilutions of 52, 62, and 76% were generated from the “100% Percoll” solution using 1X HBSS-EDTA (Thermo Fisher Scientific, 14025092, United States). The 76, 62, and 52% Percoll separation solutions were successively added to a 15 ml centrifuge tube (avoiding mixing of the three concentrations of Percoll separation solutions). The bone marrow cell suspension was overlaid on the Percoll separation layer, followed by centrifugation for 30 min at 2,800 rpm (1,420 g) at room temperature without braking. Cells were harvested from the 76 and 62% Percoll interface and washed twice with 1X HBSS buffer. 3 ml of Histopaque-1119 (Sigma-Aldrich, 11191, United States) was added to a 15-ml conical centrifuge tube, which was then overlaid with the cell suspension, followed by centrifugation for 30 min at 2,000 rpm (724 g) at room temperature without braking. The neutrophils were then collected at the Histopaque-1119 interface. The surface markers of neutrophils (Ly-6G⁺CD11b⁺) were analyzed by flow cytometry.