Fig. 4

Ang1 is released independently from NETs. Neutrophils from HC subjects were stimulated with PBS, LPS (100 nM), PMA (25 nM), or A23187 (5 μM) for 3 h to induce NETosis. Following stimulation, neutrophils were incubated with a primary unconjugated Ab (Rabbit α-human Ang1), followed by an incubation with a conjugated secondary Ab (Alexa Fluor 488 goat anti-rabbit; white). For the negative control, neutrophils were incubated only with the secondary Ab (blue boxes). Neutrophils were labeled with wheat germ agglutinin (conjugated with Alexa 647; red) and NETs were labeled with SYTOX Orange (green pseudo-color). Maximum intensity projection from acquired Z-stack were obtained by confocal microscopy (LSM 710, Carl Zeiss) using a Plan Apochromat 40x/1.3 oil DIC objective