The reaction was stopped with 2 N sulphuric acid, and the plates were read using dual wavelengths (465 and 590 nm) on a microplate reader (Spectra Max 190, Molecular Devices). selleck chemicals The cytokine concentrations were determined by comparison to a standard curve prepared using the recombinant murine cytokines (R&D Systems) that could be detected at 4–10 pg/mL. The cytokine

concentrations were expressed as the amount of induced cytokine in picograms per 106 macrophages. The production of LXA4 and 15-epi-LXA4 was determined from cell-free supernatants acidified with 1 N HCl to pH 3.4–3.6 and passed slowly through an octadecylsilyl silica column (C18 Sep-Pak® column, Waters® Corporation, USA) that had been pre-washed

with 10 ml of absolute ethanol and 10 ml of water. After activating the column with 10 ml of water, 2 ml of absolute ethanol and 2 ml of water, the eicosanoids were eluted from the column with 1 ml of water, 1 ml of ether and 2 ml of methyl formate, and the samples were dried under a stream of nitrogen. LXA4 and 15-epi-LXA4 concentrations Osimertinib in vivo were determined using an ELISA kit (Neogen Corporation, USA). The sensitivity of the assays was 2 ng/mL. Statistical analyses of the differences between the groups were performed according to Glantz (1997) using GraphPad InStat software, version 3.01 (GraphPad Software Inc., San Diego, CA, USA). A one-way analysis of variance followed by Tukey’s test was used for multiple comparisons (all pairs of groups) of the values from

the assays using the Boc-2 antagonist. To analyse the data from the other assays, a one-way analysis of variance was used, followed by Bonferroni’s test for multiple comparisons against a single control or by an unpaired Student t-test to compare two groups. Differences with P < 0.05 were considered statistically significant. The results are presented as the mean values ± standard error of Aspartate means. Treatment with CTX for 2 h increased the amount of H2O2 liberated by the macrophage monocultures (60%) and by macrophages co-cultivated with tumour cells (41%) at 24 h of incubation (Fig. 1A). After this period, this oxygen reactive molecule was not detected in either culture. As shown in Fig. 1B, pre-treatment with CTX stimulated the NO production of macrophage monolayers (38%) and of macrophages co-cultivated with tumour cells (29%) at 48 h of incubation. The LLC-WRC 256 cell cultures produced very low levels of both reactive molecules (data not shown). Interestingly, the co-cultures of control macrophages with the tumour cells exhibited a marked reduction of H2O2 liberation (29%, Fig. 1A) and NO production (20%, Fig. 1B) compared to the control macrophages, suggesting that the tumour cells exerted a suppressor activity on macrophage function.