Time-dependent interactions of oxidant-sensitive fluoroprobes with inhibitors of cellular metabolism.

SUMMARY: We tested three oxidant sensitive fluoroprobes (dihydrorhodamine [DHR], 2',7'-dichlorodihydrofluorescein [H(2)DCF], and dihydroethidium [DHE]) for interactions with three inhibitors of mitochondrial electron transport. DHR, H(2)DCF, and DHE produced large time-dependent increases in fluorescence in a cell-free medium that contained either of the site III inhibitors antimycin (A) and 2-heptyl-4-hydroxy-quinoline-N-oxide but minimal increases in medium that contained another site III inhibitor, myxothiazol (Mx). The interactions between A and each of the fluoroprobes occurred at concentrations of agent/probe that are frequently used in experiments designed to investigate cellular oxidant production. To define more effectively the nature of these agent/probe interactions, we determined the oxygen dependence of the interactions between A and each probe. The A/H(2)DCF and A/DHR interactions either were highly oxygen-dependent or exhibited a small degree of oxygen dependence, respectively, whereas the A/DHE interaction was oxygen-independent. Finally, we determined multiple ways to reduce the impact of the agent/probe interaction on data acquisition. The addition of either fetal bovine serum (10%) or albumin (5%) to the media abolished the A/DHR and A/H(2)DCF interactions. Shifting the excitation wavelength of DHE (from 470 to 530 nm) reduced measurement of the A/DHE interaction while preserving measurement of the intracellular signal. Collectively, these results emphasize the importance of testing for interactions between agents and probes, because these interactions can interfere with the accurate interpretation of experimental results. In addition, the methods presented for circumventing these interactions may be applicable to other experiments in which agent/probe interactions are an obstacle to accurate interpretation of the experimental results.