Neutrophils were isolated from patients and HDs’ blood by density centrifugation over Dextran-Ficoll as described by Nauseef et al [34 (link)]. As neutrophils could be activated by the isolation method, CD11b and CD62L expression were evaluated in healthy neutrophils (n = 5) from buffy coat (800 x g for 15 min at room temperature (RT) with the brake off centrifugation) and after dextran sedimentation followed Ficoll-Hypaque density gradient centrifugation. Both neutrophil activation markers were measured by flow cytometry (single-laser FACScalibur cytometer, BD Biosciences, San Jose, CA, USA) using anti-human CD11b PE (BD Biosciences) and anti-human CD62L FITC (eBioscience, San Diego, CA, USA). Non-statistically significant difference in CD11b and CD62L expression was found between buffy coat and the isolation method (Fig. S1 ).
Peripheral blood mononuclear cells (PBMCs) from HDs´ whole blood were separated by Ficoll gradient centrifugation (StemCell Technology, Oslo, Norway).
Purity of the populations was assessed by flow cytometry (single-laser FACScalibur cytometer, BD Biosciences) by analyzing the size and complexity (forward and size scatters). The purity was routinely ≥95% for both neutrophils and PBMCs.
Peripheral blood mononuclear cells (PBMCs) from HDs´ whole blood were separated by Ficoll gradient centrifugation (StemCell Technology, Oslo, Norway).
Purity of the populations was assessed by flow cytometry (single-laser FACScalibur cytometer, BD Biosciences) by analyzing the size and complexity (forward and size scatters). The purity was routinely ≥95% for both neutrophils and PBMCs.