Isolation and Characterization of Neutrophils

To separate cells from plasma, 1.8 mL of citrated blood was centrifuged for 10 min at 160 g, RT. Subsequently, platelet-reach plasma was discarded, and the remaining cells were suspended in 4 mL of phosphate-buffered saline (PBS, Sigma Aldrich, St. Louis, MO, USA) and processed as described before [33 (link)]. Briefly, the cell suspension was layered over 2 mL of Histopaque 1077 (Sigma Aldrich, St. Louis, MO, USA) and centrifuged (420 g, 30 min, RT). Next, the resulting cell pellet was suspended in 4 mL of 1% polyvinyl alcohol (Avantor, Radnov, PA, USA.) and the tube was left to stand upright for 20 min, RT to let the erythrocytes sediment. Next, the upper-layer containing neutrophils and the alcohol was collected and centrifuged for 7 min at 235 g, RT. The remaining erythrocytes were removed by hypotonic lysis and the granulocytes were washed twice with PBS. The cells were suspended in culture medium (RPMI 1640, Sigma Aldrich, St. Louis, MO, USA). Experiments were performed in cells with >90% granulocyte content and 85% viability as assessed with a Cytomics FC 500 Beckman Coulter flow cytometer (Beckman Coulter, Brea, USA) by staining with anti-CD33-FITC and anti-CD15-PE antibodies (both from Beckman Coulter, Brea, CA, USA) and trypan blue dye exclusion assay.