These wells were filled with 100 μL of PRS, to which was added 10 μL of a H2O2 solution, resulting in a final concentration of H2O2 ranging from 5 to 40 μM. The subsequent rows contained 100 μL of PRS without (basal H2O2 production) or with phorbol myristate acetate Y27632 (100 ng). After 60 min of incubation at 37 °C, the reaction was stopped by the addition of 10 μL of a 1 N NaOH solution. The hydrogen peroxide-dependent phenol red oxidation

was spectrophotometrically measured at 620 nm using a Titertek Multiscan apparatus. The concentration of H2O2 was calculated from the absorbance measurements and expressed as nanomoles of H2O2 per 2 × 105 cells. To determine the nitric oxide production, nitrite was measured in the supernatants of cultures or co-cultures based on the method described by Ding et al. (1988). At the end of the culture period, 50 or 100 μL of the supernatant was removed and incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylene diamine dihydrochloride, 2.5% H3PO4) at room temperature for 10 min. The absorbance was determined using a Titertek Multiscan apparatus at 550 nm. The nitrite concentration was determined by using sodium nitrite as the standard. Cell-free medium contained 0.2–0.3 nmol of NO2−/well; LBH589 research buy this value was determined in each experiment and subtracted from that obtained with cells. The proliferation of tumour cells was assessed

using the 3-(4,5 dimethylthiazol-2-thiazyl)-2,5-diphenyl-tetrazolium 4-Aminobutyrate aminotransferase bromide (MTT, Amresco®) assay, based on the method described by Mossmann (1983) and Zhong et al. (2008). Cultured and co-cultured macrophages were maintained in RPMI 1640 culture medium at 37 °C in an environment of 5% CO2 for 48 h. After this period, 30 μL of MTT solution (5 mg/mL) was added, and the cultures were incubated for 3 h at 37 °C. During the incubation, living cells convert the tetrazolium component of the dye solution into formazan crystals. The formazan crystals were dissolved by adding 100 μL of PBS containing 10% SDS and 0.01 N

HCl and incubating the mixture for 18 h at 37 °C in 5% CO2. The absorbance was read on a multiwell scanning spectrophotometer (ELISA reader) at 570 nm. The number of cells was estimated by comparison to a standard curve prepared using known numbers of fresh live cells added to the plates immediately before staining. The cytokines present in the supernatants of the cell cultures were quantified using an ELISA. Briefly, ELISA plates (Immuno Maxisorp; Nunc, NJ) were coated with mouse anti-rat monoclonal or polyclonal antibodies against IL1-β, TNF-α and IL-6 (R&D Systems, Minneapolis, MN). The plates were incubated overnight at room temperature and blocked for 1 h at room temperature on a shaker before adding the samples and standards and incubating for 2 h. Biotinylated secondary antibodies were added before 2 h of incubation, and then, peroxidase-conjugated streptavidin was added before 20 min of incubation.