Polymorphprep

For RNA and protein extraction, neutrophils were isolated from EDTA-anticoagulated blood using Polymorphprep (Alere technologies, Norway; #AS1114683) by density-gradient centrifugation as previously reported (12 (link)). For migration assay, neutrophils were isolated by a magnetic-activated cell sorting method with anti-CD15 microbeads (Miltenyi Biotec, Germany; #130-046-601), as previously reported (13 (link)). The purity and viability of neutrophils were more than 95% confirmed by flow cytometer with CD15⁺ singlets.

Human blood was obtained via venipuncture from healthy donors after obtaining informed consent. The protocol was approved by the institutional review boards of Osaka University Graduate School of Dentistry (H26-E43). Human neutrophils and monocytes were prepared using Polymorphprep (Alere Technologies AS, Oslo, Norway), according to the manufacturer's instructions. Human blood was carefully layered on the Polymorphprep solution in centrifugation tubes, which were then centrifuged at 450 × g for 30 min in a swing-out rotor at 20°C. Monocyte and neutrophil fractions were transferred into tubes containing ACK buffer (0.15 M NH₄Cl, 0.01 M KHCO₃, 0.1 mM EDTA), then centrifuged, washed in phosphate-buffered saline (PBS), and resuspended in RPMI 1640 medium.

Human whole blood from healthy volunteers was collected into ethylenediaminetetraacetic acid (EDTA)-containing tube. Neutrophils were isolated by density gradient centrifugation using Polymorphprep (Alere Technologies, Germany) according to the manufacturer’s instructions. Neutrophils were re-suspended with Roswell Park Memorial Institute (RPMI) 1640 (Gibco/Life Technologies, MA, USA) without phenol red supplemented with 1% fetal bovine serum (FBS; Gibco/Life Technologies, MA, USA) for normal culture conditions. Alternatively, neutrophils were cultured under serum-free (i.e., RPMI-1640 with 0% FBS) or albumin-depleted (i.e., RPMI-1640 with 1% FBS without albumin—see “Albumin depletion from serum or plasma”) conditions. For the replenishment of albumin, bovine serum albumin (BSA; BioShop Canada Inc., Canada) dissolved in phosphate buffered saline (PBS) was freshly prepared and added to albumin-free media at a final concentration of 0.02 g per dL, matching the albumin concentration in the condition of 1% FBS. Neutrophil purity was established to be routinely >90%, as assessed by May-Grünwald Giemsa (Sigma-Aldrich, Canada) staining.

Freshly drawn whole blood (20 ml) was layered onto Polymorphprep gradient centrifugation medium (20 ml, Alere Technologies AS, Oslo, Norway) to separate granulocytes and peripheral blood mononuclear cells (PBMCs) from red blood cells by centrifugation at 500 g for 30 min at room temperature (RT). The cell suspension containing granulocytes and PBMCs (15 ml) was layered on Ficoll-Paque PLUS (30 ml, GE Healthcare, Uppsala, Sweden) and centrifuged at 400 g for 20 min at RT to separate PBMCs and granulocytes. Monocytes were isolated from PBMCs by negative depletion of non-monocytes via indirect magnetic labeling (Pan Monocyte Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). The amount of residual red blood cells and platelets in monocyte and granulocyte preparations was determined by blood cell counting (Sysmex KX-21 N, Sysmex, Neumuenster, Germany), and monocyte and granulocyte purity was additionally characterized by flow cytometry after staining with CD14-PE and CD45-PB for monocytes and with CD66b-APC for granulocytes.

Human PMNs were isolated from the heparinized whole blood of healthy volunteers and sepsis patients using Polymorphprep™ centrifugation (Alere Technologies AS, Oslo, Norway). The neutrophil population was >95 % pure, as determined by Wright–Giemsa staining.