Isolation of Neutrophils from Healthy Donors

Blood was collected from healthy volunteer donors after obtaining informed consent into sodium heparin tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands). Neutrophils were isolated using density gradient followed by erythrocyte lysis, as previously described [23 (link)]. Neutrophils were then resuspended in IMDM (Gibco; Thermo Fischer Scientific Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated (HI) fetal bovine serum (FBS; Hyclone; Thermo Fischer Scientific Inc., Waltham, MA, USA) and gentamycin (86 µg/mL; Duchefa Biochemie B.V., Haarlem, The Netherlands) and used immediately. Neutrophil purity was analyzed by flow cytometry and was always >97%.