Isolation of Polymorphonuclear Leukocytes

Blood samples were collected in EDTA-coated tubes. To isolate PMNs from whole blood samples, we applied density gradient centrifugation [31 (link)]. The blood samples were diluted with Dulbecco’s phosphate buffered saline (D-PBS; Lonza, Basel, Switzerland), gently layered on Ficoll-sodium diatrizoate (Lymfoprep, Axis-Shield PoC AS, Dundee, United Kingdom) and centrifuged (400× g, 30 min, 20 °C, without break). The bottom layer with PMNs and red blood cells was first diluted with D-PBS and then gently mixed with 6% Dextran (Sigma-Aldrich, St. Louis, MO, USA) in Milli-Q water to induce red blood cell aggregation and sedimentation. After an incubation period of 30 min at 37 °C, PMNs were isolated. We performed two washing steps with D-PBS and lysed residual red blood cells by a hypotonic shock [31 (link)]. After two additional washing steps, PMNs (>95% pure) were ready for use.