The results were expressed as pg/mL for each cytokine. Neutrophils (2 × 105 cells/50 μL) were incubated with different concentrations of BbV (1.5, 3, 6, 12.5, 25, 50 e 100 μg/mL) or RPMI (control) or PMA (500 ng/mL, positive control) for 4 and 15 h at 37 °C in a humid atmosphere (5% CO2). After centrifugation, the supernatant was used to determine NETs release accordingly to the procedure described in kit Quant-iT™ Picogreen dsDNA (Invitrogen).

Briefly, 50 μL of samples were incubated with 100 μL of PI (Quant-iT) and 50 μL of PE buffer in a 96-well dark plate. After 15 min of incubation, absorbances at 520 nm emission and 480 nm excitation were recorded and NETs release was estimated from a standard curve. The results were represented as ng/mL of DNA. Means and S.E.M. of all data were obtained and compared by one-way ANOVA, followed RG7204 ic50 by a Tukey test with significance probability levels less than 0.05. In order to investigate the effect of BbV on neutrophil

function we isolated these cells using a density gradient. The purity of the isolated neutrophils obtained with the density gradient was 98.5% as determined by flow cytometry using the pan-granulocyte marker CD66b (Mannoni et al., 1982) and by Panotic staining Epacadostat concentration of cytospin preparations (Inserted). We used an MTT assay to test the toxicity of BbV on isolated human neutrophils. To this end, the effect of 2 and 15 h of incubation on several concentrations of BbV was investigated. As shown in Fig. 1, incubation of BbV at all concentrations used did not affect human neutrophil viability in comparison with control cells incubated with culture medium alone at all-time intervals. This finding is evidence that BbV is not toxic to human neutrophils for these periods of time and at these concentrations. To verify the ability of BbV to induce the production of hydrogen peroxide by human neutrophils, the cells were incubated with the venom in

non-cytotoxic concentrations or PMA (positive control) or RPMI (negative control). As shown in Fig. 2 incubation of neutrophils IKBKE at concentrations from 6.2 up to 100 μg/mL resulted in a significant increase in hydrogen peroxide production. These findings demonstrated the ability of BbV to stimulate human neutrophils to produce hydrogen peroxide. To investigate the ability of BbV to induce the release of PGE2 by human neutrophils, the concentration of this lipid mediator in the supernatant of neutrophils incubated with BbV (1.5, 3.1, 6.2, 12.5, 25, 50 and 100 μg/mL) or PMA (positive control; 500 ng/mL) or RPMI (negative control) was measured. Incubation of neutrophils with BbV for 4 h induced a significant increase in the basal levels of PGE2 in the supernatant of all concentrations examined in comparison to controls (Fig. 3) suggesting that PGE2 has a role in acute inflammation inducing the activation of neutrophils.