FIGURE 3.

Reductive conversion of Janus green B (JG-B) to diethylsafranine is specifically observed only in the presence of mitochondria (and not in the presence of post-mitochondrial supernatant), and the amount of formed diethylsafranine correlates significantly with the increase in the amount of mitochondria. (A) Absorption spectra of JG-B alone, incubated with mitochondria, or with post-mitochondrial supernatant are shown. Conversion of JG-B to diethylsafranine occurs only in the presence of mitochondria, observed as a peak at 550 nm (marked with the arrow). (B) Reductive cleavage of JG-B to diethylsafranine, measured as the absorbance ratio 550 nm/595 nm increases with the increase in the amount of mitochondria and follows a linear trend. (C) No formation of diethylsafranine was observed when JG-B was incubated with mitochondria-depleted post-mitochondrial supernatant, even up to 2000 µg of protein. (D) Binding and conversion of JG-B to diethylsafranine, assessed after mitochondrial pelleting and removal of free (unbound) JG-B, follows a linear trend with increasing amounts of mitochondria. Data is presented as mean ± standard error of the mean [SEM] (n = 4).