Fig. 6

a Eosinophils were more prone to produce extracellular traps compared to neutrophils from the same donor (n = 7 healthy blood donors). Eosinophils and neutrophils were purified from peripheral blood using density centrifugation followed by MACS eosinophil isolation kit. The cells that bound to the magnetic beads are regarded as neutrophils and the ones that did not as eosinophils (Additional file 2). The cells were seeded on poly L-lysin coated cover slides, stimulated with PBS, PMA, TNF or C5a for 3 h and stained using DAPI and anti-nucleosome antibodies. The percentage of cells that had released neutrophil/eosinophil extra cellular traps (NETs/EETs) were counted under the microscope. The Wilcoxon matched pairs signed rank test was used to calculate the level of significance. b Shows a representative immunofluorescence experiment: DAPI is shown in blue and nucleosomes in red. Eosinophils are shown in i, iii and v and neutrophils in ii, iv and vi. In i and ii the cells were incubated with PBS, in iii and iv with C5a and in v and vi with PMA for 3 h. Cells stimulated with TNFα did not differ visually from the cells stimulated with C5a. c Eosinophils from anti-neutrophil cytoplasmic antibodies associated vasculitides (AAV) patients produced more EETs after stimulation with C5a and ANCA IgG compared to stimulation with C5a and IgG purified from healthy blood donors (HBD). This difference was not seen in eosinophils purified from HBD. Eosinophils were purified from peripheral blood using Eosinophils MACS Xpress kit and put on poly L-lysin coated cover slides. They were then primed for 15 min with C5a or PBS followed by the addition of purified IgG from ANCA positive patients or IgG from HBD and a 3 h incubation. They were thereafter stained using DAPI and anti-nucleosome antibodies. The percentage of cells that had undergone NET/EETosis were counted under the microscope. The two-sided Mann-Whitney test was used to calculate the level of significance. d Shows eosinophils from a GPA patient purified with MACSXpress kit that were incubated for 3 h at 37 °C and 5% CO_2. In i) the cells were incubated with PBS, ii) with PMA, iii) cells that were primed with C5a for 15 min followed by purified IgG from a healthy blood donor and iv) cells that were primed with C5a for 15 min followed by purified IgG from an ANCA positive patient. DNA is visualized with DAPI (blue) and nucleosomes visualized with an anti-nucleosome antibody labelled with Alexafluor 594 (red). The EETs do generally not contain many granular proteins as the granules are released intact during EETosis (Additional file 5)