Fig. 2. Contribution of mitochondria to superoxide generation. (A) Ax2 cells were starved in shaking suspension for 6 hours at a density of 2x10⁷ cells/ml, then resuspended at 10⁸ cells/ml, and 0.5 mM XTT and the indicated concentration (µM) of rotenone added. After 10 minutes the absorbance at 470 nm was measured. The data are the means ± s.e.m. of three experiments. (B) Exponentially growing Ax2 cells were washed and resuspended in phosphate buffer at a density of 2x10⁶ cells/ml. Duplicate samples were treated with rotenone or DMSO carrier for 10 minutes and then for a further 10 minutes with 10 µg/ml rhodamine 123. Cells were washed briefly three times in phosphate buffer and the degree of rhodamine sequestration determined as the relative fluorescence measured using a spectrofluorimeter. One experiment typical of three is shown. Fluorescence microscopy confirmed that rhodamine 123 accumulated in punctate, filamentous structures as expected for mitochondria in cells not treated with rotenone (Matsuyama and Maeda, 1995), but no such staining was detectable in cells treated with 2 µM rotenone (data not shown).