A rapid method for the evaluation of compounds with mitochondria-protective properties.

Mitochondrial dysfunction has been implicated in a number of neurodegenerative diseases, such as ischemia and Parkinson's disease. We present here a method that allows the rapid quantification of interventions, aimed at inhibiting the effect of mitochondrial membrane potential uncouplers, based on the ratioing properties of the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), by using currently available 96-well fluorescent plate readers. A method is presented for evaluation of cross-talk between the two excitation/emission channels. Further characterization of the probe shows that the effect of plasma membrane potential changes on JC-1 fluorescence ratio are negligible, but that the signal is very sensitive to pH. One of the most exciting applications is the possibility to perform end-point measurements, thanks to the ratioing properties of the probe. The system is tested in different culture types with different mitochondrial uncouplers. As an example of a quantitative evaluation, we show that flunarizine is able to inhibit, dose-dependently, FCCP mediated JC-1 signal increase. The procedure is simple and allows for the fast screening of mitochondria-protective compounds.