Fig. 3.
Representative TMRE images from vehicle-treated and 300 nm staurosporine-treated rat hippocampal cultures.A, E, Phase-contrast (PC) images. B, F, Baseline TMRE fluorescence. Images were taken after loading cells with 100 nm TMRE.C, G, Peak TMRE fluorescence after treatment with 0.1 μm FCCP. The higher fluorescence of staurosporine-treated cells is clearly visible. D,H, Corresponding traces of the two experiments. After loading with TMRE, baseline fluorescence was monitored for 5 min. Note the stability of the values, indicating that the dye distribution was fully equilibrated. At 5 min, the cultures were exposed to 0.1 μm FCCP, which depolarizes the mitochondria, resulting in a characteristic increase in fluorescence (“unquenching”) as the dye concentration equilibrates with that outside the mitochondria. The mitochondria of both control and staurosporine-treated cells could be depolarized with FCCP. After 5 min of FCCP exposure, the buffer was exchanged, and cells were allowed to recover ΔΨm. Scale bar, 20 μm.