Increased NETosis in patients with MPN vs healthy donors. (A-B) Representative fluorescent imaging of NETs confirming NETosis in primary human neutrophils after 4 hours of exposure to (A) dimethyl sulfoxide and (B) ionomycin 4 µM (original magnification ×40). Antibody staining labeled citrullinated histone H3 red (Cy5, red) and human MPO green (Cy3, yellow); DNA was counterstained with diamidino-2-phenylindole (DAPI) (blue). (C) Induced NETosis rates measured in primary neutrophils from 103 individual MPN patients and 27 healthy donors (HD) by ELISA. (D) Quantification of free DNA in cell culture supernatant from NETosis induction by 4 μM ionomycin demonstrated increased DNA levels in MPN patient samples compared with those from healthy donors. Data are presented as x-fold of vehicle controls. Boxplots show the 25% and 75% quartiles and the median; overlayed dot plots indicate individual measurements.