The cells were washed and resuspended in PBS containing 0.1% paraformaldehyde. The cells cytometric analyses (104 events per data acquisition file) were performed with FACScalibur using Cell Quest software (Becton NADPH-oxidase inhibitor Dickinson). All flow cytometry experiments were performed in triplicate of three independent experiments. Soluble E-selectin and IL-8 released in the HUVECs culture supernatants were measured in the first 6 h of treatment with jararhagin (200 nM) or LPS (1 ng/mL) by ELISA, according to the manufacturer’s instructions (Duo Set® ELISA Development Systems – R&D Systems). The concentrations of E-selectin and IL-8 were calculated by interpolation of the

regression curve of known amounts of recombinant proteins as provided in the Duo Set® System and the results were reported as pg/mL of cell culture supernatant. The data were presented as mean ± standard deviation (SD) for each group. Differences between groups were assessed by Student-t test; Two-way ANOVA and the Bonferroni multiple comparison test using the GraphPad Prism Software v 4.0 (Inc., San Diego, USA). A p value < 0.05

was considered as statistically significant for the microarray and real-time experiments. The cell viability and cell detachment experiments were analyzed with p value < 0.01. This experiment was performed in order to establish the minimal dose of jararhagin that would induce cell adhesion, small decrease in cell viability and with the capacity to activate human vascular endothelial cells. We can observe in Fig. 1 that HUVECs treated with different doses of jararhagin did not detach from the

click here substrate (gelatin 0.1%) during the first 6 h (Fig. 1A). However after 24 and 48 h, a significant cell detachment was observed for all doses of jararhagin (Fig. 1A). Moreover, a decrease of cell viability was observed after 24 h of jararhagin treatment increasing according to the dose (100, 200 or 400 nM) and this effect was more accentuated after 48 h (Fig. 1B). Thus we can conclude that the effects of jararhagin on cell detachment and viability are dose and time-dependent. Considering previous study performed Cyclooxygenase (COX) by our group, showing that 800 nM of jararhagin on HUVECs induces 50% of cell detachment from the substrate and 12% of cells undergo apoptosis during the first 24 h (Baldo et al., 2008), we used 200 nM of jararhagin in our experiments as a low toxic and sub-apoptotic dose, inducing a partial endothelial cell detachment during the first 24 h of treatment. The LPS (1 μg/mL) was used as a positive control of endothelial cell activation and did not induce any cell detachment from the substrate or cell toxicity, at all time intervals analyzed. To gain a global perspective from the nature of the changes in HUVECs gene expression induced by jararhagin treatment (200 nM at 24 h) a microarray experiment was performed using the Affymetrix HgU133 A probe set. The GeneChip data obtained were analyzed using Ingenuity Pathway Analysis Software.