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

Mitochondrial dysfunction has been implicated in a number of neurodegenerat ive diseases, such as ischemia and Parkinson's disease. We present here a m ethod that allows the rapid quantification of interventions, aimed at inhib iting the effect of mitochondrial membrane potential uncouplers, based on t he ratioing properties of the fluorescent probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), by using currently a vailable 96-well fluorescent plate readers. A method is presented for evalu ation of cross-talk between the two excitation/emission channels. Further c haracterization of the probe shows that the effect of plasma membrane poten tial changes on JC-I fluorescence ratio are negligible, but that the signal is very sensitive to pH. One of the most exciting applications is the poss ibility to perform end-point measurements, thanks to the ratioing propertie s of the probe. The system is tested in different culture types with differ ent mitochondrial uncouplers. As an example of a quantitative evaluation, w e show that flunarizine is able to inhibit, dose-dependently, FCCP mediated JC-l signal increase. The procedure is simple and allows for the fast scre ening of mitochondria-protective compounds (C) 1999 Elsevier Science B.V. A ll rights reserved.