Fig. 3.

Dose, kinetic and functional inhibition assays of N. caninum tachyzoites-triggered NET formation in dolphins. PMN were incubated with tachyzoites, zymosan (1 mg/ml, positive control) or plain medium (negative control) at different ratios (a; PMN: tachyzoites = 1:1, 1:2, 1:3) and time periods (b; 30, 60 and 90 min). To prove the DNA nature of NETs, the samples were treated with DNase I (a; 15 min). Moreover, cetacean PMN cells were pre-treated with NOX-inhibitor (b; DPI, 10 μM) for 30 min prior to N. caninum stimulation (1:3 ratio; 90 min). After incubation, all samples were analyzed for extracellular DNA by quantifying Pico Green^®-derived fluorescence intensities. Each condition was performed in duplicates. Geometric means of three PMN donors. Differences were regarded as significant at a level of p < 0.05 (*) and p < 0.01 (**).