TNF- induced cytochrome c release from mitochondria. CEM cells (A through C) and CEM/VLB100 cells (D through F) were incubated with or without 250 U/mL TNF- for 3 hours, labeled with MitoTracker, fixed, permeabilized, stained with native anti-cytochrome c antibody, and finally counterstained with DAPI as described in Materials and Methods. The stained cells were examined by fluorescence microscopy. Cytochrome c antibody was visualized with a fluorescent-conjugated anti-mouse IgG and assigned the color green, whereas mitochondria labeled with MitoTracker were assigned the color red. In the control cells (A and D) and CEM cells treated with TNF- for 3 hours (B), red and green images were merged; overlapping red and green pixels appears orange/yellow. CEM/VLB100 cells treated with TNF- for 3 hours displayed separation of the green (cytochrome c) from the mitochondria (red) with appearance in the cytosol (E and F). C and F are cells similar to B and E but counterstained with the nuclear dye DAPI. Cells with condensed and/or fragmented nuclei are apoptotic cells (arrows).
