Fig. 6.

Effects of sulforaphane pretreatment on reoxygenation induced mitochondrial reactive oxygen species generation in bEnd.3 cells adapted to 18 kPa or 5 kPa O₂

bEnd.3 cells seeded in Ibidi μ-Slide 8-well chambers were cultured under 18 or 5 kPa O₂ for 5 d. Cells were subjected to hypoxia (1 kPa O₂, 1 h) and loaded with MitoSOX™ Red for 5 min before the start of 30 min reoxygenation under 18 or 5 kPa O₂, respectively. Control cells were loaded with MitoSOX™ Red during the last 30 min of an experiment. Cells were fixed with 4% paraformaldehyde and images acquired using a Nikon Diaphot microscope with a 40× objective. (A) Representative images of MitoSOX fluorescence and DAPI stained nuclei after 30 min reoxygenation and (B) quantification of MitoSOX fluorescence. (C) bEnd.3 cells were pre-treated with vehicle (0.01% DMSO) or SFN (2.5 μM) for 24 h before exposure to hypoxia (1 h) and reoxygenation under 18 or 5 kPa O₂, respectively. Representative images of MitoSOX fluorescence and DAPI stained nuclei after 30 min reoxygenation and (D–E) quantitation of MitoSOX fluorescence. Each symbol in panels B, D and E represents the mean fluorescence from at least 10 cells in a field of view, with each color denoting a different bEnd.3 experiment with at least 6 different fields of view. Data denote mean ± S.E.M., n = 18–20 fields of view in each of 3 independent bEnd.3 cell cultures, two-way ANOVA followed by Bonferroni post-hoc analysis, ****P < 0.0001. Scale bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)