For the experiments, the cells were seeded at a density of 5 × 104 cells/mL in the medium described above. The co-culture

procedure that was used was based on the method described by Hauptmann et al. (1993). Co-cultures were established in 96-well tissue culture plates to determine the hydrogen peroxide (H2O2) liberation and nitric oxide (NO) production and to assess tumour cell proliferation. LLC-WRC 256 tumour cells (1 × 104/100 μL per well) were allowed to adhere for 3 h, washed with PBS and incubated with fresh medium for 24 h. The adherent peritoneal macrophages (2 × 105) were pre-treated with CTX (0.3 μg/mL) for 2 h, washed, collected and transferred to a 96-well tissue culture plate containing 2 Χ 104 tumour cells per well. The macrophage cultures and co-cultures were maintained

selleck chemicals llc for 24 and 48 h at 37 °C in a 5% CO2 humidified atmosphere. To determine the production of IL-1β, TNF-α, IL-6, LXA4 and 15-epi-LXA4, the adherent peritoneal macrophages (5 × 105) were pre-treated with CTX (0.3 μg/mL) for 2 h, washed, collected and transferred to a 24-well plate containing LLC-WRC 256 tumour cells (5 × 104 per well) plated in fresh medium 24 h beforehand. The macrophage cultures and co-cultures were maintained for 12, 24 and 48 h at 37 °C in a 5% CO2 humidified atmosphere. This resulted in a macrophage:tumour ratio of 10:1. www.selleckchem.com/products/AZD6244.html All the experiments were performed in triplicate, with macrophages from three different donors. The concentration of CTX (0.3 μg/mL) was the same as that used in previous research (Sampaio et al., 2003, Sampaio et al., 2006a and Sampaio et al., 2006b), which did not exhibit cytotoxicity as assessed by Trypan blue exclusion and by flow cytometry for the exclusion of propidium iodide. The involvement of the

formyl peptide receptor (ALX or FPR1) in the stimulatory effect of CTX on the secretory activities of macrophages was evaluated in cells pre-treated with 100 μM of Boc-2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe, Phoenix Pharmaceutical Inc, USA), a selective antagonist of formyl peptide receptors, for 15 min at 37 °C (Scannell et al., 2007) before incubation with CTX, as described above. The production of H2O2 was measured as described by Pick et al. (1981), using phenol red. This assay is based on a horseradish peroxidase-dependent conversion of phenol red into a coloured compound by H2O2. Cobimetinib A phenol red solution (PRS) containing 140 mM NaCl; 10 mM potassium-phosphate buffer, pH 7.0; 5 mM dextrose; 0.28 mM phenol red; and 8.5 U/mL of horseradish peroxidase was used for the H2O2 determination. A final volume of 7.4 ml was obtained using Hank’s solution. After 24 h of co-culture, the supernatants were collected, and 100 μL of phenol red solution was added into each well of 96-well flat-bottomed tissue culture plates (Corning, NY), which were incubated in a humidified atmosphere at 37 °C for 1 h. Vertical row no. 1, which lacked cells, was filled with 100 μL of PRS per well.