Fig. 6. Intracellular distribution of cytochrome c in apoptotic and non-apoptotic human-derived HL-60 cells after infection with T. gondii. Cells were infected at a parasite to host ratio of 30:1 and were then treated with 5 µg/ml actinomycin D for 8 hours. The subcellular distribution of cytochrome c was determined by triple immunofluorescence staining and confocal microscopy. After fixation and permeabilization, cell preparations were stained using fluorescein-labelled dUTP to visualize DNA strand breaks (green fluorescence), a cytochrome c-specific monoclonal antibody and Cy3-conjugated secondary antibody (red fluorescence), and a Toxoplasma-specific antiserum and Cy5-conjugated secondary antibody (blue fluorescence).(A) Single optical sections from representative cells of the indicated treatments are shown. In non-apoptotic cells (i.e. those without signs of DNA strand breaks), cytochrome c was granularly distributed indicating a mitochondrial localization (thick arrows), while this molecule was homogenously distributed in apoptotic cells indicating translocation into the cytoplasm (arrowheads). Parasite-positive cells showed no signs of apoptosis and this was correlated with a granular (i.e. mitochondrial) distribution of cytochrome c (thin arrows). (B) 10 optical sections for each cell preparation were taken at intervals of 0.5 µm and were superimposed. A fluorescence intensity profile of the cytochrome c labelling was determined for selected cells as indicated by the straight line in the overlay micrographs. The lowercase letters below the intensity profiles refer to the cells or parts thereof for which the subcellular distribution of cytochrome c has been determined.