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Short answer: TMRE (tetramethylrhodamine ethyl ester) is a cell-permeable, cationic fluorescent dye that accumulates in the mitochondrial matrix according to mitochondrial membrane potential, so its signal is used as a readout of that potential. MitoSOX is a different tool: a mitochondria-targeted hydroethidine analog used to probe superoxide, and its red fluorescence alone is not specific for superoxide.
This page is general reference for researchers and lab staff. It is not a protocol and gives no step-by-step procedure; follow the instructions supplied with your specific kit or reagent.
What TMRE is
The Cayman Chemical TMRE Mitochondrial Membrane Potential Assay Kit insert (Item 701310) describes TMRE as a cell-permeable, cationic dye that accumulates in the mitochondrial matrix based on mitochondrial membrane potential. The same kit supplies FCCP, a mitochondrial uncoupler, as a positive control for depolarization. The kit is described as usable with plate readers, fluorescence microscopes and flow cytometers; for plate-reader use the insert lists excitation and emission of 530 and 580 nm.
For background on why membrane potential matters, see the CASRAI guide on mitochondria and their function.
What MitoSOX is, and why it is often confused with TMRE
Both reagents are sold as “mitochondrial dyes”, which is why they are often grouped together, but they answer different questions. Michalski et al. (Free Radical Biology and Medicine, 2013) use the names “Mito-HE” and “MitoSOX Red” for a mitochondria-targeted analog of hydroethidine. Kalyanaraman et al. (Biochimica et Biophysica Acta, 2014) report that superoxide reacts with hydroethidine to form the diagnostic product 2-hydroxyethidium, and not from hydrogen peroxide, hydroxyl radical or peroxynitrite. Because the fluorescence spectra of ethidium and 2-hydroxyethidium overlap, they conclude that fluorescence techniques using the red fluorescence are not suitable for detecting and measuring superoxide in cells with hydroethidine or structurally analogous probes, naming MitoSOX Red explicitly. HPLC-based assays that separate 2-hydroxyethidium from other products are the approach they describe, and they state that fluorescence-microscopic methods should be avoided for this purpose.
TMRE and MitoSOX compared
| Question | TMRE | MitoSOX Red |
|---|---|---|
| What kind of molecule | Cell-permeable, cationic dye (Cayman insert 701310) | Mitochondria-targeted hydroethidine analog (Michalski et al., 2013) |
| What the signal is used to read | Mitochondrial membrane potential (Cayman insert 701310) | Intended as a superoxide probe; red fluorescence alone does not specifically identify superoxide (Kalyanaraman et al., 2014) |
| Control named in the sources | FCCP uncoupler as a positive control for depolarization (Cayman insert 701310) | HPLC separation of 2-hydroxyethidium from other products (Kalyanaraman et al., 2014) |
Controls and checks that apply to a TMRE experiment
The Cayman insert supports a short checklist of design points:
- Include a depolarization control. FCCP is supplied as the positive control; a signal that does not fall with an uncoupler is not behaving as a membrane-potential readout.
- Optimize dye concentration for each cell line. The insert says concentrations in the 5 to 250 nM range should be examined, and that signal rises with dye concentration until a self-quenching concentration is reached. Too much dye is listed in troubleshooting as a cause of high background and self-quenching.
- Check signal against background. The insert gives an optimal signal of at least three times the background measured without cells.
- Protect from light. The dye is light sensitive and the insert calls for incubation in the dark.
The insert also reports its own data for HepG2 cells, with no observable self-quenching up to 500 nM at sub-confluent density, which illustrates why the useful range depends on cell line and density rather than being a fixed number.
Where this matters for lab decisions
Because the three instrument formats named above (plate reader, fluorescence microscope, flow cytometer) all work with the kit, the choice of readout depends on what your lab already runs. Whichever you use, culture quality is part of any live-cell fluorescence readout; see mycoplasma testing methods for one common check. For another fluorescence-based cell imaging approach, see Cell Painting.
Common misunderstandings
- “TMRE and MitoSOX both measure mitochondrial health.” They report different things: membrane potential versus an oxidant-related probe signal.
- “A red MitoSOX signal means superoxide.” Per Kalyanaraman et al. (2014), red fluorescence alone cannot specifically identify superoxide because of overlapping spectra.
- “More TMRE gives more signal.” Only up to a self-quenching concentration (Cayman insert 701310).
Sources
- Cayman Chemical. TMRE Mitochondrial Membrane Potential Assay Kit, Item 701310 insert. cdn.caymanchem.com/cdn/insert/701310.pdf. Read 2026-11-08.
- Kalyanaraman B, et al. Biochim Biophys Acta 2014;1840(2):739-744. doi:10.1016/j.bbagen.2013.05.008. PubMed 23668959. Read 2026-11-08.
- Michalski R, et al. Free Radic Biol Med 2013;54:135-147. PubMed 23051008. Read 2026-11-08.
