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Mitochondria are a double-membrane-bound organelles that generate most of the cell’s energy. Here we describe a range of probes that are available to identify the mitochondria structure in live and fixed cells. The uptake of most of these dyes are dependent on mitochondria membrane potential enabling researchers to evaluate mitochondrial activity, localization, and abundance. Fluorescent fusion proteins are also available to identify the mitochondria and are independent of mitochondria membrane potential.
Mitochondria are found in most eukaryotic cells, where they make up as much as 10% of the cell volume. The mitochondria structure is a double-membrane that consists of an outer membrane and an inner membrane called a cristae that extends into the inner matrix [1]. Mitochondria are pleomorphic organelles, with structural variations depending on cell type, cell cycle stage, and intracellular metabolic state. The key function of mitochondria is energy production through oxidative phosphorylation and lipid oxidation. However, mitochondria are involved in various other metabolic functions including urea production, steroid biogenesis, and calcium homeostasis.
MitoTracker Green FM | MitoTracker Orange CMTMRos | MitoTracker Red CMXRos | MitoTracker Red FM | MitoTracker Deep Red FM | |
Readout | Fluorescent staining of mitochondria | ||||
Target | MitoTracker Dyes selectively label mitochondria based on membrane potential | ||||
Common filter set | FITC | TRITC | Texas Red | Cy5 | Cy5 |
Labels | MitoTracker Green | MitoTracker Orange | MitoTracker Red | MitoTracker Red FM | MitoTracker Deep Red |
Ex/Em (nm) | 490/516 | 554/576 | 579/599 | 581/644 | 644/665 |
Signal-to-noise ratio | |||||
Photostability | |||||
Multiplexing | Yes | Yes | Yes | Yes | Yes |
Live cells | Yes | Yes | Yes | Yes | Yes |
Fixed cells | No | No | No | No | No |
Fixable | No | Yes | Yes | No | Yes |
Platform | Imaging | Imaging | Imaging | Imaging | Imaging |
Format | 20 x 50 μg | 20 x 50 μg | 20 x 50 μg | 20 x 50 μg | 20 x 50 μg |
Cat. No. | M7514 | M7510 | M7512 | M22425 | M22426 |
Readout |
Expression of fluorescent fusion protein
| |
Target |
Labels pyruvate dehydrogenase in mitochondria
| |
Common filter set | FITC | TRITC |
Labels | GFP | RFP |
Ex/Em (nm) | 488/520 | 555/584 |
Signal-to-noise ratio | ||
Photostability | ||
Multiplexing |
Yes
|
Yes
|
Live cells |
Yes
|
Yes
|
Fixed cells |
No
|
No
|
Fixable |
Yes
|
Yes
|
Platform |
Imaging
|
Imaging
|
Format |
1 mL
|
1 mL
|
Cat. No. |
MitoTracker Green FM | MitoTracker Orange CMTMRos | MitoTracker Red CMXRos | MitoTracker Red FM | MitoTracker Deep Red FM | |
Readout | Fluorescent staining of mitochondria | ||||
Target | MitoTracker Dyes selectively label mitochondria based on membrane potential | ||||
Common filter set | FITC | TRITC | Texas Red | Cy5 | Cy5 |
Labels | MitoTracker Green | MitoTracker Orange | MitoTracker Red | MitoTracker Red FM | MitoTracker Deep Red |
Ex/Em (nm) | 490/516 | 554/576 | 579/599 | 581/644 | 644/665 |
Signal-to-noise ratio | |||||
Photostability | |||||
Multiplexing | Yes | Yes | Yes | Yes | Yes |
Live cells | Yes | Yes | Yes | Yes | Yes |
Fixed cells | No | No | No | No | No |
Fixable | No | Yes | Yes | No | Yes |
Platform | Imaging | Imaging | Imaging | Imaging | Imaging |
Format | 20 x 50 μg | 20 x 50 μg | 20 x 50 μg | 20 x 50 μg | 20 x 50 μg |
Cat. No. | M7514 | M7510 | M7512 | M22425 | M22426 |
Readout |
Expression of fluorescent fusion protein
| |
Target |
Labels pyruvate dehydrogenase in mitochondria
| |
Common filter set | FITC | TRITC |
Labels | GFP | RFP |
Ex/Em (nm) | 488/520 | 555/584 |
Signal-to-noise ratio | ||
Photostability | ||
Multiplexing |
Yes
|
Yes
|
Live cells |
Yes
|
Yes
|
Fixed cells |
No
|
No
|
Fixable |
Yes
|
Yes
|
Platform |
Imaging
|
Imaging
|
Format |
1 mL
|
1 mL
|
Cat. No. |
MitoTracker probes are cell-permeant mitochondria stains that contain a mildly thiol-reactive chloromethyl moiety. Following incubation, MitoTracker dyes passively diffuse across the plasma membrane and accumulate in the mitochondria of live cells. These dyes are offered in a range of wavelengths that can all be used for mitochondrial localization in multicolor experiments (Figures 1–3).
Signal retention of the MitoTracker probes is variable after cell fixation. MitoTracker Green and MitoTracker Red lose signal, while MitoTracker Orange CMTMRos, MitoTracker Red CMXRos, and MitoTracker Deep Red are well retained. Cells stained with MitoTracker Deep Red can be subsequently permeabilized and still retain its staining pattern.
MitoTracker Orange CM-H2TMRos and MitoTracker Red CM-H2XRos are additional mitochondrial stains. These probes do not fluoresce until they enter live cells, where they are oxidized to become fluorescent and then are sequestered in the mitochondria.
Figure 1. MitoTracker Green FM stained cells. U2OS cells labeled using LysoTracker Blue DND-22, MitoTracker Green FM, and Tubulin Tracker Deep Red show multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 60X Super Apochromat Oil objective using DAPI, GFP, and Cy5 EVOS light cubes.
Figure 2. MitoTracker Orange CMTMRos stained cells.Human Cardiac Arterial Smooth Muscle (HCASM) primary cells labeled using NucBlue Live ReadyProbes Reagent, MitoTracker Orange CMTMRos, and Tubulin Tracker Deep Red show multiplexing capability and staining specificity. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 100X Super Apochromat Oil objective using DAPI, RFP, and Cy5 EVOS light cubes.
Figure 3. MitoTracker Red CMXRos stained cells. HeLa cells labeled using Tubulin Tracker Green Variety Pack and MitoTracker Red CMXRos show multiplexing capability and uniformity of staining. Cells were imaged in Gibco HBSS buffer containing calcium and magnesium, supplemented with 1X Probenecid solution. Images were generated using an EVOS FL Auto 2 Imaging System with an Olympus 20X Super Apochromat objective using GFP and Texas Red EVOS light cubes.
CellLight fluorescent fusion proteins can be used to stain mitochondria and follow the dynamics of mitochondria behavior in live cells independent of membrane potential. Thus, they can be used in combination with MitoTracker dyes to investigate relationships between mitochondria morphology and membrane potential. CellLight Mitochondria-GFP (Figure 4) and CellLight Mitochondria-RFP (Figure 5) are ready-to-use constructs that use the leader sequence of E1 alpha-pyruvate dehydrogenase (3.1 kDa) to target mitochondria.
Introducing CellLight fluorescent fusion proteins involves a simple transfection step, using the BacMam technology, and they work like cell stains with minimal toxicity or chemical disruption. These mitochondrial fusion proteins are compatible with other fluorescent probes for multiplex analysis in live cells, or after formaldehyde fixation for colocalization studies.
Learn more about these and other CellLight fluorescent reagents
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For Research Use Only. Not for use in diagnostic procedures.