S monovette 9 ml

Blood samples were collected by peripheral venous puncture the day before surgery during routine preoperative blood draw. Soluble ICAM-1 and CEA levels were determined in samples from the same blood draw. Blood was drawn in S-Monovette 9 ml, Clotting Activator/Serum (Sarstedt, Nümbrecht, Germany, ref. 02.1063) and was allowed to clot for 30 min. After centrifugation at 2500×g for 10 min at room temperature, the supernatant was collected in aliquots and frozen at − 80 °C until analysis. Soluble ICAM-1 was measured using the human ICAM-1/CD54 non-allele-specific Quantikine ELISA kit (Cat. no. SCIM00, R&D Systems, Inc. Minneapolis, USA) following the manufacturer’s instructions. Every blood sample was measured in duplicates. The mean value of sICAM-1 was calculated and used for further analysis.

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.

PBMCs, isolated from freshly taken EDTA blood, were used as reference for the experiments. Briefly, venous blood was collected with a butterfly needle into 9 mL tubes (S-Monovette 9 mL, Sarstedt, Sarstedt, Germany). In total 7.5 mL blood were layered onto 5 mL of lymphocyte separation medium (PAA Laboratories, Pasching, Austria) and centrifuged for 20 min at 1000× g without breaks. The PBMC layer was transferred into a fresh tube and washed twice with PBS. The cells were counted and seeded in a density of 5 × 10⁵ cells/mL. For experiments the PBMCs were seeded in a density of 5 × 10⁵ cells/mL and cultured at 37 °C (5% CO₂, humidified atmosphere).