The absence of live extracellular bacteria was ensured after subculture on agar plates. At each time point, cells were lysed by 0.1% Triton X-100, and viable intracellular bacteria were counted by plating
serial dilutions of lysates on blood agar plates. Peripheral blood mononuclear cells (106 mL−1) were stimulated with live bacterial cells for 45 min at a ratio of 1 : 10, as in preliminary experiments, this ratio was proved to be the most efficient in cytokine production. Afterwards, extracellular bacteria were lysed by lysostaphin Obeticholic Acid purchase (Sigma), and medium was replaced by CM supplemented with antibiotics. Cells were incubated for selected time points. Each experiment was carried out with mononuclear cells isolated from a single donor and performed in triplicate. Results are based on at least three experiments from different
donors. LPS (10 ng mL−1) was used as a positive control, and PBMCs without stimulants, bacteria or LPS were used to assess spontaneous levels of cytokine secretion (negative control). Supernatants were collected, and the levels of TNFα, IL-1β, IL-6, IL-8, GM-CSF and IL-12p40 were measured by Human Cytokine Multiplex Immunoassay kit manufactured by Linco Research Inc. using Luminex® xMAP™ technology, whereas the levels of IL-12p70, IFN-γ and IL-13 were measured by High Sensitivity Human Cytokine Multiplex Immunoassay Akt inhibitor kit. A five-parameter regression formula was used to calculate
the cytokine concentrations in samples from standard curves. MDMs (2 × 105 mL−1) were stimulated with bacteria at a ratio 1 : 10 for 45 min, extracellular bacteria were lysed by lysostaphin and medium was replaced by CM supplemented with antibiotics. Cells were incubated for additional 12, 24 and 48 h. Supernatants were collected, and the levels of TNFa, IL-1b, IL-6, IL-12p40 and IL-12p70 were assessed. Statistical analysis was performed using spss 17 statistical package (SPSS Inc.). Differences in PIA concentration between biofilm, planktonic and control cells and extracts were assessed by anova test followed by pairwise comparisons. Differential adhesion of biofilm and planktonic cells Oxalosuccinic acid on human MDMs was evaluated using paired t-test. anova test was applied to assess differences in cytokine induction between reference and clinical strains for both parameters (planktonic and biofilm phase). No difference was found (anova P > 0.05); therefore, data analysis was performed after comparing results from all strains. Differences in cytokine concentrations between planktonic and biofilm phase were evaluated using paired t-test. Two-way anova was used to evaluate differences in intracellular survival between biofilm and planktonic phase cells. Statistical significance was set at α = 0.05.