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Mitochondrial membrane potential probes and the proton gradient: a practical usage guide
Seth W. Perry1,2, John P. Norman3, Justin Barbieri4, Edward B. Brown1, and Harris A. Gelbard2,4, 5
1Department of Biomedical Engineering, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
2Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
3Graduate Program in Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
4Center for Neural Development and Disease, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
5Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
BioTechniques, Vol. 50, No. 2, February 2011, pp. 98–115
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Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.

In recent years, fluorescent dyes for measuring the mitochondrial membrane potential (Δψm) have become commonly used tools for monitoring changes in this important physiologic mitochondrial parameter as it relates to cells’ capacity to generate ATP by oxidative phosphorylation. As such, the Δψm is a key indicator of cell health or injury. As a class, these dyes are typically lipophilic cationic compounds that equilibrate across membranes in a Nernstian fashion, thus accumulating into the mitochondrial membrane matrix space in inverse proportion to Δψm (1-4). A more negative (i.e., more polarized) Δψm will accumulate more dye, and vice versa. A handful of dyes can be used for this purpose, each with their own strengths and weaknesses (Table 1) (for review, see Reference 2). For best usage, all of these dyes require strict attention to technical details and controls to ensure appropriate interpretation of dye behavior as related to Δψm (2,5,6). Herein, we aim to complement other excellent, high-level reviews detailing theoretical and applied aspects of these dyes in life sciences research (e.g., References 1,2,3, and references therein) by offering a practical guide that lays out in one place— especially for laboratories using these tools for the first time—the range of controls and supporting experiments required to ensure meaningful interpretation of results obtained with these dyes.

Why is Δψm important?

As the energy power-plants of the cell, mitochondria generate ATP by utilizing the proton electrochemical gradient potential, or electrochemical proton motive force (Δp), generated by serial reduction of electrons through the respiratory electron transport chain (ETC). The reductive transfer of electrons through ETC protein complexes I–IV in the inner mitochondria membrane provides the energy to drive protons against their concentration gradient across the inner mitochondrial membrane (out of the mitochondrial cytoplasm). This results in a net accumulation of H+ outside the membrane, which then flows back into the mitochondria through the ATP-generating F1/F0 ATP-synthase (Complex V), thus producing ATP and completing the ETC. The total force driving protons into the mitochondria (i.e., Δp), is a combination of both the mitochondrial membrane potential (Δψm, a charge or electrical gradient) and the mitochondrial pH gradient (ΔpHm, an H+ chemical or concentration gradient). Using a simplified Nernst factor for the second term, Δp can be represented at 37°C by the equation: Δp (mV) = Δψm – 60ΔpHm (2,7,8). Using approximate physiological values of Δψm = 150 mV and ΔpHm = –0.5 units (mitochondrial matrix is alkaline), this equates to Δp = 150 – 60(–0.5) = 180 mV (mitochondrial matrix is negative) (2,7,9). Typical Δp values range 180–220 mV, with Δψm typically accounting for 150–180 mV of this value, and ΔpHm of 0.5–1.0 units contributing the remaining 30–60 mV per the Nernst factor (2,7,10,11). This equation and the later case study (Box 2) also help illustrate an important distinction: the probes described herein are simply measuring the charge gradient Δψm across the inner mitochondrial membrane; they do not and cannot specifically measure the mitochondrial proton gradient, ΔpHm. To assess this parameter, other tools are required (see Section 10, and the case study outlined in Box 2).

Together, these factors help regulate mitochondrial control over energy metabolism, intracellular ion homeostasis, and cell death in eukaryotic cells. While Δp provides the bioenergetic driving force for and regulates ATP production, the Δψm component of Δp provides the charge gradient required for mitochondrial Ca2+ sequestration, and regulates reactive oxygen species (ROS) production, and thus is also a central regulator of cell health (2,12). During cellular stress, Δψm may in turn be altered by dysregulation of intracellular ionic charges [e.g., Ca2+ (2,7,12) or K+ (13)], consequently changing Δp and thus ATP production. When ionic fluxes surpass the ability of mitochondria to buffer these changes, ultimately Δp, Δψm, and/or ΔpHm may collapse, leading to a failure of ATP production and bioenergetic stress. Several extensive and excellent reviews explore these mitochondrial bioenergetics in more detail (7,14,15).

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