Fig. 5

ROS analyses with DA-DCFH and MitoSOX probes. a Analysis of reactive oxygen species (ROS) by flow citometry of A549 cells (panels a and b) and myofibroblasts (panels c and d). Cells were pretreated for 2 h with 5 nmol/L vitamin D and then treated for 24 h with bleomycin (12,5 μg/mL) and/or 5 nmol/L vitamin D. The cultures were then washed twice with Hank’s balanced salt solution (HBSS) and incubated for 1 h with HBSS. After that, the cultures were incubated with DA-DCFH (20 μM) in HBSS for 1 h, washed and trypsinized. Trypsin was neutralized with HBSS-FBS 2% and the cells were collected and analyzed immediately by flow cytometry. The treatments with MitoSOX were carried-out as for the DA-DCFH probe. Cells were incubated for 1 h with 5 μM MitoSOX in HBSS and then processed for flow cytometry. Controls for ROS induction employed in these experiments consisted of treatment for 1 h with 50 μM terbutyl hydroperoxide (terB). The results presented in the graphs are means of the fluorescence intensity ± SEM. ANOVA result a (P < 0,001), ANOVA results b, c and d (P < 0,0001). Significance of the analysis post-test is indicated in the figure as *, P < 0.05; **, P < 0.01; and ***, P < 0.001