A new method for testing cell ageing using two mitochondria specific fluorescent probes.

Cell culture techniques have considerably improved our understanding of the numerous changes related to aging. For instance, murine lymphocytes obtained from animals older than 6 months progressively lose their, in vitro, proliferative capacity. Numerous studies have shown that this loss is due to changes in the mitochondrial compartment such as reduction in the transmembrane potential and/or membrane mass. Using two mitochondria specific probes with a potential-dependent (Rhodamine 123) or independent (Nonyl Acridine Orange) uptake, we found that the decline in the respiratory activity in the mouse occurred approximately 6 months prior to the decrease in mitochondrial membrane mass. The analysis of the Rh 123/NAO fluorescence ratio measured in splenocytes obtained from mice aged more than 6 months, showed that there was a linear loss of respiratory efficiency per unit of mitochondrial membrane mass. Moreover, cells with a ratio of less than 0.85 were incapable of proliferating and remained quiescent. The time separating the infection points of the two dye uptake curves might provide informations about the regulation and coordination of nuclear and/or mitochondrial genomes. Moreover, the ratio between the two fluorescent probes, in particular during the linear phase, may also have a predictive value.