Confocal microscopy analysis of the effect of pHPP on production of mitochondrial superoxide anion in cultured cells.

<p>EA.hy 926 cells were grown on coverslips in complete DMEM +10% FBS (<b>A</b>) or without FBS (<b>B</b>) (for 4 hours) either in the absence and in the presence of 10 mM pHPP followed by addition of the mt-O₂^.− fluorescent probe MitoSox (0.5 µM, 15 min incubation). The pictures are optical fields under each condition as imaged by laser scanning confocal microscopy and are representative of two independent experiments yielding similar outcomes. White bar: 50 µm. The insets show selected enlargements rendered to highlight the intracellular compartmentalization of the fluorescent signal. (<b>C</b>) Histogram showing the quantitative analysis of the intracellular fluorescent signal along with the statistical significance of the differences; bars indicate mean values ± SD from 5 randomly selected optical fields/experiment taken under each condition (each optical field contained ≈20 cells). See under Materials and Methods for further details.