Use of Potentiometric Fluorophores in the Measurement of Mitochondrial Reactive Oxygen Species

Abstract

Mitochondrial reactive oxygen species (ROS) are implicated in signal transduction, inflammation, neurodegenerative disorders, and normal aging. Net ROS release by isolated brain mitochondria derived from a mixture of neurons and glia is readily quantified using fluorescent dyes. Measuring intracellular ROS in intact neurons or glia and assigning the origin to mitochondria are far more difficult. In recent years, the protonmotive force crucial to mitochondrial function has been exploited to target a variety of compounds to the highly negative mitochondrial matrix using the lipophilic triphenylphosphonium cation (TPP+) as a “delivery” conjugate. Among these, MitoSOX Red, also called mito-hydroethidine or mitodihydroethidium, is prevalently used for mitochondrial ROS estimation. Although the TPP+ moiety of MitoSOX enables the many-fold accumulation of ROS-sensitive hydroethidine in the mitochondrial matrix, the membrane potential sensitivity conferred by TPP+ creates a daunting set of challenges not often considered in the application of this dye. This chapter provides recommendations and cautionary notes on the use of potentiometric fluorescent indicators for the approximation of mitochondrial ROS in live neurons, with principles that can be extrapolated to non-neuronal cell types. It is concluded that mitochondrial membrane potential changes render accurate estimation of mitochondrial ROS using MitoSOX difficult to impossible. Consequently, knowledge of mitochondrial membrane potential is essential to the application of potentiometric fluorophores for the measurement of intramitochondrial ROS.

Bioenergetic Analysis of Single Pancreatic β-Cells Indicates an Impaired Metabolic Signature in Type 2 Diabetic Subjects

“…Levels of mitochondrial reactive oxygen species (ROS) were measured as the MitoSOX to MTDR fluorescence ratio (see Supplemental Figure 3) to compensate for possible differences in cell geometry and altered membrane potential dependent loading of MitoSOX (24). Figure 2E indicates significantly higher ROS levels in T2D ␤-cells than in normal ␤-cells, when an outlier normal measurement is rejected.…”

Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV

“…Importantly, MitoSOX redistributes out of mitochondria in response to stimuli that decrease mitochondrial membrane potential [21, 22]. This renders estimation of mitochondrial ROS based on cellular MitoSOX fluorescence semi-quantitative at best.…”

“…This is because factors other than mitochondrial ROS affect the total cellular fluorescent signal once mitochondria are depolarized. We found that cellular MitoSOX fluorescence was enhanced when oligomycin was used to prevent ATP synthase reversal from maintaining mitochondrial membrane potential in the presence of an electron transport chain inhibitor [22]. This observation suggests that increased nucleic acid availability (i.e., cytoplasmic RNA and nuclear DNA) following efflux of MitoSOX from mitochondria may modify the fluorescent signal independently of changes in mitochondrial ROS generation (see [22] for a detailed discussion).…”

“…We found that cellular MitoSOX fluorescence was enhanced when oligomycin was used to prevent ATP synthase reversal from maintaining mitochondrial membrane potential in the presence of an electron transport chain inhibitor [22]. This observation suggests that increased nucleic acid availability (i.e., cytoplasmic RNA and nuclear DNA) following efflux of MitoSOX from mitochondria may modify the fluorescent signal independently of changes in mitochondrial ROS generation (see [22] for a detailed discussion). Thus, great caution should be applied when using MitoSOX to make inferences about mitochondria-derived ROS following drug treatments or disease states with altered mitochondrial bioenergetic properties.…”

“…Even in the absence of treatment, concentration- and time-dependent redistribution of MitoSOX dye to the nucleus has been observed by multiple investigators [12, 22, 23]. This suggests that matrix accumulation of MitoSOX oxidation products dissipates mitochondrial membrane potential, thereby decreasing charge-based dye sequestration.…”