Fluorescence measurement of absolute mitochondrial membrane potential in adherent cultured cells

Abstract

An assay of in situ mitochondrial membrane potential (Δψm) within live cells matched with respirometry is an invaluable tool for kinetic analysis of the electron transport chain. All known Δψm measurement techniques (fluorescence, voltammetric or isotopic) rely on the Nernstian distribution of lipophilic cations, such as the fluorescent tetramethylrhodamine methyl ester (TMRM). The accumulation of these cations in cells is not only a function of Δψm, but also of the plasma membrane potential (Δψp), the matrix to cell volume ratio, and its activity coefficients and binding in the matrix and the cytosol. When comparing different cell types or genetic manipulations, these parameters are not expected to be the same. We have developed a fluorescence microscopy based technology for absolute determination of Δψm in millivolts and to monitor its changes in time in live adherent cells. Δψp is followed with a lipophilic anionic plasma membrane potential indicator (PMPI). The Δψp-dependent distribution of the probes through the plasma membrane is modeled by electrostatic barrier limited diffusion, and a solution of this model is used to deconvolute Δψp in time from changes of PMPI fluorescence intensity. Then, Δψm is determined in millivolts by deconvoluting TMRM fluorescence taking in account the slow, Δψp-dependent redistribution and its Nernstian behavior. The electrostatic barrier model was verified by voltage clamp in perforated whole cell mode, combined with fluorescence imaging. The volume and activity coefficient ratios are determined in separate, purely fluorescence microscopic methods that minimize the uncertainty of the suboptical matrix to mitochondrion volume ratio. The absolute value of Δψm is calculated from the efflux kinetics of TMRM following an acute and complete mitochondrial depolarization. Ultimately, time courses of fluorescence intensity are converted into millivolts with the minimal requirement of complete depolarization of the Δψm and then of the Δψp at the end of the assay. The calculations are performed by standard Excel functions. The technique has been validated on rat cortical neuronal cultures, yielding Δψm values in accordance to earlier radioisotope studies. Δψm determinations have been performed on INS-1E, HEK and PC12 cultures, and on human embryonic stem cells and derivatives.