, San Diego, CA) and incubated
overnight at 4°C. After the removal of the capture antibody solution, 100 μl of PBS supplemented with 2% BSA (blocking buffer) were added to each well and incubated at room temperature for 2 h. Next, cytokine standards and samples diluted in blocking buffer supplemented with 0.05% Tween-20 were added to the respective wells and incubated overnight at 4°C. At the end of the incubation, after three washings steps with PBS supplemented with 0.05% Tween-20, 100 μl of biotinylated antibody solution were added to the wells and incubated for 2 h at room temperature. After three washing steps, streptavidin–horseradish peroxidase conjugate (1:2000 dilution; Biolegend) were then added to the wells and incubated for 1 h at room temperature. Finally, after washing, 100 μl of 63 mM Na2HPO4, 29 mM citric acid AZD9291 in vivo (pH 6.0) containing 0.66 mg ml-1 o-phenylenediamine/HCl
and 0.05% hydrogen peroxide were dispensed into each well, and the wells were allowed to develop. The absorbance was read at 415 nm and the cytokine concentrations were calculated using standard curves and expressed as pg ml-1. Cell viability, redox status and phase 2 enzyme activity Lactate dehydrogenase Ku-0059436 mw (LDH) in spent media was measured [26] to determine the effects of the different treatments on eukaryotic cell viability. Release of total thiols [GSHtot, GSH + glutathione disulfide (GSSG)], GSH and GSSG concentrations in cytosolic extracts were quantified using the 5,5′-dithionitrobenzoic acid (DTNB)-GSSG reductase recycling method [27]. Upon normalization to protein
content, intracellular GSH and GSSG were expressed as nmoles mg-1 min-1. The extracellular thiol level was expressed as nmoles min-1. NQO1 and GST activities were measured in cytosolic extracts as previously described [28], and the obtained values were normalized to the protein content and expressed as nmoles 1-chloro-2,4-dinitrobenzene (CDNB) mg-1 min-1 and nmoles NAD mg-1 min-1, respectively. Statistical analysis Statistical significance was determined by t-test or ANOVA using the GraphPad PRISM 4.0 software (GraphPad Avelestat (AZD9668) Software, Inc., La Jolla, CA). A P-value of 0.05 or less was considered to be significant. Results Probiotic properties of L. gasseri OLL2809 and L13-Ia L. gasseri OLL2809 and L13-Ia have been isolated from human intestine and raw bovine milk, respectively, and their properties have previously been reported [22, 23]. To further assess these strains’ probiotic features, we focused on their antimicrobial activity. Table 1 shows the inhibition halos produced by L13-Ia and OLL2809 against four pathogenic bacterial strains. The supernatants of both strains were found to be effective against all tested pathogens without significant differences in their inhibitory activity. This indicated that the two strains of L.