Lympholyte poly cell separation medium

PBMCs were isolated from fresh EDTA blood. Venous blood was collected in EDTA tubes (S-Monovette, Sarstedt, Sarstedt, Germany) and directly used for density gradient centrifugation. A total of 6 mL of blood was carefully layered on 6 mL of Lympholyte-poly Cell Separation Medium (Cedarlane, Burlington, ON, Canada). Samples were centrifuged for 35 min at 500 g without a break at room temperature. The PBMC (upper) layer was transferred to a new tube. After washing twice with PBS, the cells were counted and seeded at a concentration of 5 × 10⁵ cells/mL in an RPMI 1640 medium with 2% autologous plasma [28 (link)]. Experiments were performed at 37 °C (5% CO₂, humidified atmosphere).

Neutrophils were isolated freshly from blood taken from 15 healthy volunteers. Venous blood was taken with a butterfly needle into EDTA-tubes (S-Monovette 9 mL, Sarstedt, Germany) and directly used for density gradient centrifugation. 6 mL blood was carefully layered on 6 mL of Lympholyte poly cell separation medium (Cedarlane, Burlington, Ontario, Canada). Samples were centrifuged for 35 min at 500 g without break at room temperature. The plasma and peripheral blood mononuclear cell (PBMC) layers were discarded and the polymorphonuclear cell (PMN) layer carefully taken with a pipette and transferred to a 15 mL tube. PMN layer was washed twice with PBS (12 mL, centrifugation at 450 g, 10 min, room temperature without break) and taken up in RPMI medium (RPMI-1640 without phenol red, Sigma-Aldrich, Munich, Germany). Cells were counted by Trypan Blue exclusion method in a Neubauer counting chamber, without counting of residual erythrocytes. Cells were prepared to a density of 1x10⁶ cells/mL.

Venous blood was freshly collected in EDTA-tubes (S-Monovette 9 mL, Sarstedt, Germany). Neutrophil isolation was performed as previously described [53 (link)]. A total of 6 mL blood was layered on 6 mL of Lympholyte poly-cell separation medium (Cedarlane, Burlington, ON, Canada), followed by centrifugation at 500× g and 40 min at room temperature, without break. The plasma and PBMC layers were discarded, and the PMN layer was carefully collected into a fresh 15 mL tube. Cells were washed twice with 12 mL PBS and centrifuged at 400× g for 10 min at room temperature, at acceleration 5 and deceleration 4. The neutrophil pellet was resuspended in RPMI medium without phenol red (Sigma-Aldrich, Darmstadt, Germany). Cells were counted using the Trypan Blue exclusion method and a Neubauer counting chamber, omitting erythrocytes from the count.