S monovettes edta

All experiments with human neutrophils were approved by the Ethics Committee of the University Medical Center (UMG) Göttingen (protocol number: 29/1/17). Neutrophils were isolated from fresh venous blood of healthy donors. Beforehand, all donors were fully informed about possible risks, and their informed consent was obtained in writing. The consent could be withdrawn at any time during the study. Blood was collected in S-Monovettes EDTA (7.5 ml, Sarstedt), and neutrophils were isolated according to previously published protocols based on histopaque 1119 (Sigma Aldrich) as well as Percoll (GE Healthcare) density gradients (17 (link), 42 (link)). Neutrophils were resuspended in HBSS^−Ca2+/Mg2+ and further diluted in the desired medium as described in the appropriate methods sections and figure legends. Purity of the cell preparation was >95% as assessed by cytospin (Cytospin 2 centrifuge, Shanson) and Diff Quick staining (Medion, Diagnostics).

Human neutrophils were isolated from venous blood of healthy donors. For all human studies, a pool of 15 healthy donors was available. For all experiments with human neutrophils, blood from at least 3 different donors from this pool was collected to isolate neutrophils. This study was carried out in accordance with the recommendations and with the approval of the Medical Ethics Committee of the University Medical Center Göttingen (UMG), protocol number 29/1/17 with written informed consent from all subjects and in accordance with the Declaration of Helsinki.
The isolation was performed under sterile conditions based on previously published protocols (31 (link)) using gradient centrifugation. In brief, fresh blood of healthy donors was collected in S-Monovettes EDTA (7.5 ml, Sarstedt) and immediately layered in a ratio of 1 to 1 on top of Histopaque 1119 (Sigma Aldrich). After centrifugation and washing with HBSS (without Ca²⁺/Mg²⁺) (Lonza) cells were separated a second time on a gradient consisting of 65, 70, 75, 80, and 85% of 10:1 diluted Percoll (GE Healthcare). Then, cells were washed and resuspended in 1 ml HBSS without Ca²⁺/Mg²⁺. Cellular identity and a purity of the isolated cells of >95% was confirmed by cytospin (Cytospin 2 Zentrifuge, Shanson) followed by Diff Quick staining (Medion Diagnostics).

All experiments with human neutrophils were approved by the Ethics Committee of the University Medical Center Göttingen (protocol number: 29/1/17). Neutrophils were isolated from fresh venous blood of healthy donors. Beforehand, all donors were fully informed about possible risks, and the informed consent was obtained in writing; consent could be withdrawn at any time during the study. Blood was received in S-Monovettes EDTA (7.5 mL, Sarstedt, Sarstedt, Germany) and neutrophils isolated according to previously published standard protocols (12 (link),41 (link)). Neutrophils were resuspended in 1 mL HBSS^-Ca2+/Mg2+. Cells were counted and further diluted at the required concentration for the following procedures in RPMI 1640 containing 10 mM HEPES (Roth, Watertown, NY, USA) and 0.5% human serum albumin (Sigma-Aldrich, Burlington, MA, USA). Purity of the isolation was assessed by a cytospin assay (Cytospin 2 Zentrifuge, Shandon, Runcorn, UK) and Diff Quick staining (Medion Diagnostics, West Bengal, India). Cell purity was always greater than 98%.