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Production of reactive oxygen species (ROS) in eosinophils. Cell aggregates were excluded based on forward scatter height and area properties, then granulocytes were gated based on their forward and side scatter. Eosinophils (in red) were defined as Siglec-8+ granulocytes. It was possible to select eosinophils also by their forward and side scatter characteristics. Intracellular production of ROS was measured as the geometric median fluorescence intensity in eosinophils (red) as a comparison typical graphs of ROS production in neutrophils are shown to the left (green). The two top histograms show unstimulated (PBS) cells and the bottom two histograms show cells activated with phorbol-12-myristate-13-acetate (PMA). (PDF 141 kb)

The percentage of eosinophils of polymorphonuclear leukocytes (PMN) and basophils of the leukocytes populations is shown when the patients are divided into active and inactive (A and B) or into GPA or MPA (C and D). Patients with active disease had lower levels of both eosinophils and basophils but no difference were seen comparing GPA and MPA. Kruskal-Wallis test and Dunn’s multiple comparisons test was used to calculate the level of significance between the three groups. Values are reported as median ± IQR. (PDF 86 kb)

Cytospin preparations of purified neutrophils and eosinophils stained with May-Grünwald Giemsa. In the first set of experiment neutrophils and eosinophils were isolated using Histopaque 1119 (Sigma) followed by Percoll (GE Healthcare) and thereafter the eosinophils were separated from the granulocytes using MACS Eosinophil Isolation Kit (Miltenyi Biotech). The cells that were removed from during the eosinophil purification step were regarded as neutrophils (A) and the ones that remained as eosinophils (B). In the second part of the experiment eosinophils were purified using the MACSXpress® Eosinophil Isolation kit (Miltenyi Biotech) (C). (PDF 199 kb)

Light microscopy picture of two eosinophils that has formed EETs after incubation with PMA for 3 h at 37 °C and 5%CO2. The white arrow indicates the web formed by the DNA and the black arrows indicate the intact granules that remains around the plasma membrane remnants. (PDF 263 kb)

The level of surface expression on eosinophils of A CD16, B CD64, C CD35, D CD193, E CD62L, F CD88, G Siglec-8, H CD11b and I CD11c was measured in healthy blood donors (HBD) and compared to anti-neutrophil cytoplasmic antibodies associated vasculitides (AAV) patients, divided into GPA and MPA patients, using flow cytometry and reported as geometric mean fluorescence intensity (MFI). Kruskal-Wallis test and Dunn’s multiple comparisons test was used to calculate the level of significance between the three groups. Values are reported as median ± IQR. No difference was seen between the GPA and MPA groups. (PDF 95 kb)

Gating strategies. (DOCX 188 kb)