Ficoll gradient centrifugation

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.

PBMCs from whole blood were separated by Ficoll gradient centrifugation (StemCell Technology, Oslo, Norway). Thereafter, the separation of monocytes and lymphocytes from the mononuclear layer was performed by the immunomagnetic depletion of non-monocytes, using commercially available kits (Monocyte Isolation kit II, Miltentyi Biotec Bergisch Gladbach, Germany). Purity of the fractions was assessed by flow cytometry (single-laser FACScalibur cytometer, BD Biosciences, San Jose, CA, USA), analyzing the size and complexity of each population (forward and size scatters). The purity was routinely ≥95% for both lymphocytes and monocytes.