Effect of oligomycin (a) and STS (b–e) on cellular TMRE uptake in D283 medulloblastoma cells and simulation of TMRE fluorescence changes according to Ward et al. (2000). The dotted linesrepresent the time lapse of total cellular fluorescence in percentage of baseline after equilibration with 10 nm(a–c) or 100 nm (d, e) TMRE. After equilibration of the TMRE signal, cells were exposed to 5 μm oligomycin (a) or 3 μm STS (b–e). The onset of treatment is indicated by the arrows on the left. After the respective treatments, mitochondria were depolarized by the addition of 2 μm FCCP plus 5 μm oligomycin (arrows on the right). Simulations based on the Nernst equation were calculated with immediate changes in ΔΨM (solid lines in a,b, and d; left arrow,hyperpolarization from −150 to −190 mV) or ΔΨP(solid lines in c and e;left arrow, hyperpolarization from −60 to −90 mV). In each case, this was followed by a simulation of mitochondrial depolarization after addition of FCCP plus oligomycin (right arrows, depolarization from −190 to 0 mV). Note that the time lapse of the oligomycin and STS treatments have the same shape. Simulations based on changes in ΔΨP are less convincing, and the peak after FCCP plus oligomycin in cells loaded with 100 nm TMRE in d does not occur ine (solid lines). Traces are means from all cells within the microscope field in a typical experiment (n = 18–26 cells). The experiments were performed in duplicate (10 nm TMRE experiments) and triplicate (100 nm TMRE experiment) with similar results.
