Lymphocytes (5 × 105/well) were acutely treated (3 h) with 0.3 mM of the Z-VAD-FMK solubility dmso fatty acid mixture added or not by 2 μM of ASTA in the absence and presence of phorbol myristate acetate (PMA; 20 ng/well) used as a ROS inducer. After 3 h the absorbance was measured at 620 nm to evaluate H2O2 concentration (compared to a standard curve). Dihydroethidium (DHE) is a florescence probe and was used to measure the intracellular superoxide anion production. Once inside the cell, DHE is rapidly oxidized to ethidium (a red fluorescent compound) by superoxide with minor collaboration of other ROS. Lymphocytes (5 × 105/well)

were incubated with 5 μM DHE for 15 min at room temperature in the dark. At the beginning of the assay control cells were stimulated with PMA (20 ng/well) and 0.3 mM of the FA mixture added or not by 2 μM of ASTA. Cells were incubated in the dark at room temperature for additional 30 min. DPI (diphenylene iodonium 10 μM), an inhibitor of NADPH oxidase (Chen et al., 2007), was used to investigate if superoxide anion production occurred through NADPH-oxidase activation. Sodium

azide (SA – 400 μM) was used as a mitochondrial inhibitor. Afterwards, fluorescence was analyzed in a microplate reader (Tecan, Salzburg, Veliparib datasheet Austria) (wavelengths of excitation and emission were 396 and 590 nm, respectively). The probe DCFH-DA was primarily used as an indicator of the production of H2O2 (Keston and Brandt, 1965) but is also described as being oxidized by other ROS such as HO , ROO , NO and peroxynitrite (Crow, 1997 and Wang and Joseph, 1999). The cells (5 × 105/well) were preloaded with DCFH-DA (5 μM) by incubation in culture medium for 30 min. DCFH-DA is cleaved intracellularly by non specific esterase and turns into high fluorescent 2,7-dichlorofluoroscein (DCF) upon oxidation by ROS. After the loading

period, cells were treated with FA with or without ASTA at 2 μM and cultured for 18 h. The experiments were conducted in the presence and absence of PMA (20 ng/well). After the culture period, cells were centrifuged and resuspended in 300 μL of Tyrode’s buffer and the fluorescence was monitored in spectrofluorimeter Tecan (Salzburg, Austria) with excitation at 485 nm and emission at 530 nm. The results of this Clomifene experiment were expressed as relative units of fluorescence. Nitric oxide production was performed according to Ding et al. (1988) through nitrite ( NO2-) determination. Nitric oxide (NO ) is rapidly converted into NO2- in aqueous solutions and, therefore, the total NO2- concentration can be used as a stoichometric indicator of NO production in culture. Lymphocytes (5 × 105/well) were cultured with 0.3 mM of the FA mixture with or without 2 μM of ASTA and LPS (10 μg/well) for 4 h. EGTA (ethylene glycol tetraacetic acid, 500 μM) was used as a calcium quelator and therefore to discard NO production by constitutive calcium-dependent NOS.