Figure 3.

Dirofilaria immitis-induced dose- and viability- and diphenyleneiodonium (DPI)-independent neutrophil extracellular trap (NET) formation. (A) D. immitis microfilariae (MF; 100) were cocultured with canine polymorphonuclear neutrophils (PMN) for 180 min. For NADPH oxidase inhibition, DPI pre-treatment was used. To resolve NET formation, DNase I was added to coculture. (B) Canine PMN were cocultured with vital or heat-inactivated microfilariae (MF-HI). (C) Vital and heat-inactivated D. immitis L3 were exposed to canine PMN. Sytox Orange-derived fluorescence intensities were analyzed by spectrofluorometric analysis at an excitation wavelength of 547 nm and emission wavelength 570 nm using an automated plate monochrome reader. As negative control, PMN in plain medium were used. PMN stimulated with zymosan served as positive control.