Primed neutrophils in contact with VWF can form DNA extracellular traps. (A) Representative images of fluorescently labeled LPS-primed neutrophils (PMN, red) and extracellular DNA (exDNA, yellow) after perfusion (100 s⁻¹ shear rate) on VWF of different concentrations of neutrophils in 2 different brands of flow chambers (n = 2 donors per chamber brand). Dashed lines have been added to delimit flow channels and the well entrance. (B) Representative images of fluorescently labeled NETs at the chamber entrance showing total DNA (Hoechst 33342, blue), H3cit (green), and MPO (pink) staining after perfusion of neutrophils in Ibidi slides at 24 × 10⁶ cells per mL (upper panel, n = 3 donors and 9 channels) and in Cellix slides at 1.5 × 10⁶ cells per mL (lower panel, n = 2 donors and 4 channels) (C-D) Quantification of NETs after VWF challenge by fluorescence microscopy analysis. HNA-3a⁺ neutrophils were primed with TNFα (to mimic LPS activation) and incubated for 180 minutes with PBS (+TNFα) or VWF (+TNFα+VWF). DNA release was visualized after DNA staining with Hoechst 33342 (n = 9 fields per well, 3 wells per condition, 2 independent experiments) (mean ± SD; ***P < .005). (D) Representative fluorescence images from quantifications shown in panel C. Scale bars, 100 µm.