The frozen samples of culture supernatants of the infected BMDM were then thawed and immediately analyzed using Bio-Plex Pro Mouse Cytokine Assay (BioRad PX-478 cost Laboratories, Hercules, CA), following the manufacturers protocol. Standard curves for each cytokine were generated using reference cytokine concentrations supplied by the manufacturer. Nitric oxide determination Nitric oxide (NO) generation in the culture supernatants was assessed by the Griess method to measure nitrites, which are stable breakdown products of NO. Briefly, culture

supernatant was incubated with the Griess reagents I (1% sulfanilamide in 2.5% phosphoric acid) and II (0.1% naphthylenediamine in 2.5% phosphoric acid). The absorbency was read within 5 min at 550 nm and actual concentration calculated using a standard curve with serial dilutions of sodium nitrite. Detection of iNOS, ARG-1 and MR by Western blot The infected adherent cells were resuspended in lysis buffer (10% SDS, 20%

glycerol, 5% 2-mercaptoethanol, 2% bromphenol blue and 1 M Tris HCl, pH 6.8) for western blotting AZD6094 order analysis. Cell samples in the lysis buffer were harvested and equal CFTRinh-172 clinical trial amounts of proteins were electrophoresed in a 10% or 8% sodium SDS-PAGE gel under nonreducing conditions. The proteins were then transferred to nitrocellulose membrane (Amersham Hybond-ECL GE) using standard procedures. After overnight blocking with 0.5% non-fatty milk in PBS, the blots were incubated for 1 hr at room temperature with Ab against iNOS, 1:1000 (Santa Cruz Biotechnology, CA), Arg-1, 1:1000 (BD buy Idelalisib Bioscience), or MR/CD206, 1:100 (Santa Cruz Biotechnology, CA), dissolved in 0.5% non-fatty milk in PBS. The blots were then washed and incubated with peroxidase-conjugated secondary Ab, 1:8000, for

1 hr at room temperature, and the resulting membranes were developed using diaminobenzidine/H2O2 as a substrate for peroxidase. Densitometric analysis of the protein bands was performed using the software ImageJ for Windows (NIH, Bethesda, MD). The value for the control condition (untreated cells) was set as 1 and other conditions were recalculated correspondingly to allow ratio comparisons. Statistical analysis Statistical analysis was performed using the unpaired Student’s t test, one-way analysis of variance (ANOVA) and Bonferroni procedure for multiple range tests, employing Prism 4 software (GraphPad, San Diego, CA) to assess statistical significance between groups of data defining different error probabilities. A value of p < 0.05 was considered to be significant. Acknowledgements This work was supported by Fundação de Amparo a Pesquisa de Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.