Continuous monitoring of mitochondrial membrane potential in hepatocyte cell suspensions

Abstract

We report a simple fluorometric method for the continuous monitoring of mitochondrial membrane potential and cell viability in suspensions of hepatocytes exposed in vitro to cytotoxic agents. Suspensions of freshly isolated hepatocytes (10 6 cells/mL) preloaded with rhodamine 123 (Rh 123, 100 μmol/L) are transferred to a thermostatically controlled mixed cuvette to which the desired cytotoxic agent is added. Rh 123 is a cationic fluorophore that is actively accumulated by cells in direct proportion to the mitochondrial membrane potential. Cell viability was estimated by monitoring propidium iodide (PI) fluorescence. Exposure of cell suspensions to the mitochondrial uncoupling agent FCCP caused an immediate and titratable increase in Rh 123 fluorescence. Subsequent treatment with digitonin did not change Rh 123 fluorescence, suggeseting that Rh 123 equilibrates rapidly across the intact cell membrane. Likewise, treatment of hepatocyte suspensions with inhibitors of mitochondrial respiration (rotenone, cyanide, or menadione) caused an immediate increase in Rh 123 fluorescence. This was accompanied by a progressive increase in PI fluorescence, suggesting a causal relationship between mitochondrial depolarization and cell injury. In contrast, 1,4-benzoquinone caused a time-dependent and linear increase in PI fluorescence that paralleled changes in Rh 123 fluorescence. Comparing the time courses for changes in PI and Rh 123 fluorescence suggests that for benzoquinone, the depolarization of the mitochondria is a consequence rather than a cause of the cell injury. This modified procedure provides a simple and specific technique for continuously monitoring mitochondrial membrane potential and cell viability in suspensions of freshly isolated hepatocytes. The advantage is that there is no need to separate cells from the incubation medium, making it possible to record real-time changes in mitochondrial membrane potential and cell viability throughout the in vitro exposure period.