Granulocyte isolation and purification of neutrophils and eosinophils

Blood samples were processed within three hours after blood draw using a standard protocol for neutrophil isolation 36, 37. Each patient sample was accompanied by a concomitant control sample. Samples were processed via density gradient centrifugation with isotonic Biocoll with 10 mm hydroxyethyl‐piperazineethane‐sulfonic acid buffer (HEPES) (density of 1.077 g/mL, Biochrom). Red cells were lysed with lysis buffer containing ammonium chloride, potassium hydrogen carbonate, and ethylenediaminetetraacetic acid (EDTA) (PH 7.4) at 4°C, followed by washes in PBS and final suspension in medium, i.e. sterile‐filtered RPMI‐1640 (without Phenolred, endotoxin tested; Sigma‐Aldrich) + 2% human albumin (Albunorm 20%, Octapharma) unless otherwise indicated. Viability and purity of the obtained granulocytes was routinely ≥ 95% as assessed by trypan blue staining and flow cytometry, respectively. In healthy controls, contamination of granulocytes with eosinophils was usually < 7%. For selected experiments, granulocytes were further purified into neutrophils and eosinophils employing the EasySep Human Neutrophil Enrichment kit (Stemcell) and the EasySep Human Eosinophil Enrichment kit (Stemcell), generally achieving a purity ≥ 99% for neutrophils and ≥ 94% for eosinophils (Verification of cell origin see Supporting Information Fig. 10A–F). In order to highlight the differences between the preparations, we use the term granulocytes when referring to the initial isolation product and neutrophils when further purification steps were employed.