Fig. 3
Haemonchus contortus L3 induce the formation of aggNET upon exposure to bovine PMN. a: Bovine PMN were confronted with ensheathed and exsheathed H. contortus L3. NET measurement was performed using PicoGreen-derived fluorescence intensities and DNase I was applied 15 min before the end of incubation to the co-culture of PMN with both ensheathed and exsheathed larvae. b: Bovine PMN were confronted with ensheathed and exsheathed H. contortus L3 and aggNET were counted using phase-contrast microscopy based on a size larger than 50 mm diameter. As negative control, PMN in plain medium were used. PMN exposed to zymosan (1 mg/ml) served as positive control. c: Bovine PMN (n = 3, 5 × 105) were exposed to H. contortus L3 (20 larvae/sample) for 30 and 60 min at 37 °C. For the quantification of different types of NETs, fluorescence microscopy analysis was performed using an anti-histone H1, H2A/H2B, H3, H4 monoclonal antibody, [mouse clone H11-4, 1:1000, 1 h, RT], jointly with a secondary antibody (Alexa Fluor® 488 Goat Anti-Mouse IgG). Extracellular DNA was analysed via Sytox Orange® staining (S-11368, Invitrogen, 1:1000).