MitoSOX

MitoSOX (Mitochondrial Superoxide Indicators) are fluorescent probes specifically designed to detect and quantify mitochondrial superoxide (O2-) (Dikalov & Harrison, 2014). These superoxides are part of a larger family of compounds, known as reactive oxygen species (ROS), and form as byproducts of normal cellular metabolism, primarily during the process of oxidative phosphorylation in the electron transport chain. While low levels of mitochondrial ROS play important roles in cell signaling, metabolic regulation, and adaptive stress responses, uncontrolled production of superoxides can be harmful. For example, excess mitochondrial superoxide can induce irreversible oxidative modifications of proteins and cause genomic mutations. (Checa & Aran, 2020).

In order to study these processes and the role of superoxides, MitoSOX probes can be used. The principle of MitoSOX probes relies on their preferential oxidation by superoxides, which offer selectivity against other ROS or RNS (reactive nitrogen species). Given that MitoSOX compounds are cell permeable, these probes readily pass through the membrane of live cells, after which they accumulate in the mitochondria due to their positive charge. Therein, the probes then bind to DNA and, upon oxidation by superoxides, produce fluorescence. In this way, MitoSOX dyes are fluorogenic, and the fluorescence they produce can be measured using techniques such as fluorometry, flow cytometry, and fluorescence microscopy. (Kauffman et al., 2016).

Two popular versions of MitoSOX are MitoSOX Red and MitoSOX Deep Red; these two compounds differ primarily in their excitation and emission wavelengths. The former of these, MitoSOX Red, is a derivative of hydroethidine (HE) (Zielonka & Kalyanaraman, 2010). Both MitoSOX Red and HE are oxidized by superoxide to produce a fluorescent product, which can then be detected in cells. HE is a general ROS probe, staining ROS throughout the cell, while MitoSOX Red is a mitochondrial-targeted version of HE.

Although MitoSOX assays are highly valuable, they do have some limitations. One key drawback is that, while MitoSOX-based assays preferentially detect mitochondrial superoxide formation, these probes can be oxidized by other ROS, leading to non-specific activation. Another limitation is that high concentrations of MitoSOX may lead to underestimation of mitochondrial ROS production, as they can disrupt the mitochondrial electron transport chain, as well as cause the probe to diffuse out of the mitochondria into the cytosol. (Kalyanaraman, 2020).

As an alternative approach to MitoSOX assays, CellROX (e.g. CellROX Green) can be used to detect oxidative stress in live cells. This fluorogenic probe remains weakly fluorescent in its reduced state but emits bright green fluorescence upon oxidation by reactive oxygen species (ROS) (Sivapackiam et al., 2020); the fluorescence intensifies as CellROX binds to DNA. Notably, its signal remains stable even after formaldehyde fixation and detergent treatment. The key difference between MitoSOX and CellROX is their specificity: MitoSOX selectively measures mitochondrial ROS, while CellROX detects ROS in both the nucleus and mitochondria (Lee et al., 2021).This makes it a more versatile tool for assessing oxidative stress across multiple cellular compartments.

Beyond MitoSOX and CellROX, more broadly, ROS can be detected using several approaches, including fluorescent probes, colorimetric assays, immunofluorescence techniques, and immunoblotting. Fluorescent probes, such as DCFH-DA or MitoSOX, are commonly used to detect ROS in cells and tissues (Murphy et al., 2022). Colorimetric assays and immunoblotting can be used to measure ROS levels by detecting changes in specific proteins or other cellular components that have undergone oxidative modifications. Other methods, such as electron paramagnetic resonance (EPR), detect ROS by measuring unpaired electrons in free radicals. Oxidative damage can also be assessed by measuring biomolecule alterations, like lipid peroxidation or DNA damage. Additionally, the ability to use MitoSOX-based flow cytometry allows for high-throughput analysis of ROS levels in live cells.

When preparing MitoSOX for use, the first step is to dissolve the powder in dimethyl sulfoxide (DMSO) to create a stock solution. Gently vortex or pipette the solution to ensure that MitoSOX is completely dissolved. After dissolving, store the stock solution at −20°C for long-term storage (Kauffman et al., 2016), and protect it from light as MitoSOX is light-sensitive. Lastly, to maintain the stability of the solution, it is important to avoid repeated freeze-thaw cycles.

MitoSOX

Further Reading