MitoTracker

MitoTracker dyes are fluorescent probes used for staining mitochondria in live cells (Xiao et al., 2016).

Background

MitoTracker dyes are cell permeable compounds that can be added to live cells to stain mitochondria. Once added, these probes pass through the plasma membrane of viable cells, wherein they preferentially accumulate in active mitochondria, staining the organelles for downstream fluorescence imaging. The staining principle for MioTracker dyes relies on the compounds' cationic charge, which is hypothesized to be attracted to the mitochondrial membrane potential in a potential-dependent manner. This facilitates the visualization of mitochondrial structure and activity with minimal cytotoxicity. (Chazotte, 2011).

Advantages and Disadvantages

MitoTracker dyes offer several advantages over other similar compound families. Unlike conventional mitochondrial stains, such as tetramethylrhodamine (TMRM) and rhodamine 123, certain MitoTracker dyes do not easily leak out of the cell upon loss of membrane potential. These include the carbocyanine-based variants, such as MitoTracker Green FM, which include a cell-retention group in the chemical structure.

A second advantage is that MitoTracker dyes are offered in a variety of spectral wavelengths, to fit different instrumental and application needs. Common versions include: MitoTracker Green, MitoTracker Orange, MitoTracker Red CMXRos, and MitoTracker Deep Red. Given all the different options, it may be challenging to select the correct dye.

For typical applications, MitoTracker Green is a popular choice, due to the availability of green channels on fluorescence microscopes. If running a multiplex experiment, where the green channel is already dedicated to a different cellular target, MitoTracker Orange or MitoTracker Deep Red can be used to stain mitochondria in live cells. (Xiao et al., 2016, Agnello et al., 2008).

Comparing MitoTracker Green and MitoTracker Red, the main difference lies in their excitation and emission wavelengths. While both dyes stain active mitochondria, MitoTracker Green has an excitation / emission wavelength of 490 / 512 nm, while MitoTracker Red has an excitation / emission wavelength of 578 / 598 nm. (Nick, 2018).

While having distinctive advantages, MitoTracker dyes also do have some disadvantages in experimentation. One downside is that they are not fixable, for the most part. That is, during aldehyde fixation, for instance, wherein mitochondrial membrane potential is lost, the dyes will lose specificity for the organelle. To resolve this issue, alternative dyes, such as the fixable MitoLite stains have been developed, which are suitable for post-fixation analyses, such as immunocytochemistry and imaging applications. (Chazotte, B. 2011).

Alternatives to MitoTracker Dyes

As mentioned, MitoLite dyes offer a good alternative to MitoTracker dyes if fixation after staining is required. MitoLite dyes are also offered in more spectral wavelengths, with the addition of blue and near-infrared (NIR). Classical alternatives to MitoTracker include TMRM and rhodamine 123, although these do not perform as well as they are not as well retained in cells. For sensing calcium in mitochondria, rhod-2 is a valid alternative. Additionally, if the goal is to quantify mitochondrial membrane potential, rather than qualitatively staining the organelles, probes such as JC-1 and JC-10 are appropriate. Finally, in more recent research, a big spotlight has been placed on mitochondrial DNA (mtDNA) analysis, as a target of interest in studying human diseases and aging. For this application, the family of MitoDNA dyes is recommended. (Huang et al., 2007, Chazotte, B. 2011).

Storage

When provided as a lyophilized powder, MitoTracker dyes can last up to 6 months when stored properly -20°C in a dark location. Once reconstituted into a working solution, it should be used within a few weeks.

MitoTracker

Background

Advantages and Disadvantages

Alternatives to MitoTracker Dyes

Storage

Further Reading