Figure 2.

Mitochondrial membrane potential. A, Measurement of mitochondrial membrane potential (ΔΨ_m) in isolated mitochondria using a ΔΨ_m-sensitive dye, JC-1. Isolated brain mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice were subjected to flow-cytometric analysis in a buffer containing 20 mm glutamate plus 2 mm malate. A potentiometric dye, JC-1, accumulates in mitochondria. The emission fluorescence exhibited green and red, which were plotted on the x- and y-axes, respectively. Each point indicates fluorescence intensity from each mitochondrion under steady-state condition (control) and treatment with 1 μm FCCP (+FCCP) or 6.25 μm Ca²⁺ (+Ca²⁺). A negative shift in red signals along the y-axis indicated a loss of ΔΨ_m. B, Frequency distributions of red-to-green fluorescence ratio (R/G) of JC-1. Depolarized mitochondria particles were counted by defining that the R/G was <0.3 (a gray broken line indicates the threshold). A loss of ΔΨ_m was evaluated by decreased R/G. Ca²⁺ exposure increased the population of depolarized mitochondria from wild-type (WT) and mutPOLG Tg (Tg) mice. C, Quantifications of depolarized mitochondria. The population of depolarized mitochondria was averaged and compared with wild-type (WT) and mutPOLG Tg (Tg) under steady-state condition (control) and treatment with Ca²⁺ (+Ca²⁺) or FCCP (+FCCP). There were no significant differences between genotypes (n = 6) in ΔΨ_m or ΔΨ_m response under any of the conditions. Data are expressed as mean ± SEM.