Labeling and Visualization of Mitochondria Using Mitotracker

 To label mitochondria, cells are simply incubated with MitoTracker® probes, which passively diffuse across the plasma membrane and accumulate in active mitochondria. Once their mitochondria are labeled, the cells can be treated with an aldehyde-based fixative for samples that need fixation to allow further processing of the sample. Some of the MitoTracker® probes are also retained after permeabilization with some detergents during subsequent processing steps (e.g., immunocytochemistry or in situ hybridization).


MitoTracker Red CMXRos
MitoTracker Red CMXRos is a red-fluorescent dye that stains mitochondria in live cells and its accumulation is dependent upon membrane potential. The dye is well-retained after aldehyde fixation. allowing for further sample processing and immunostaining. Excitation: 579 nm, Emission: 599 nm, 
Invitrogen:  MitoTracker™ Red CMXRos M7512
Cell Sciences: MitoTracker® Red CMXRos #9082


MitoTracker Green FM
MitoTracker Green FM is recommended for live-cell imaging only; fixation with aldehydes or alcohols will inhibit staining. Excitation: 490 nm, Emission: 516 nm, Molecular Weight: 671.88 g/mol
Invitrogen:  MitoTracker™ Green FM M7514
Cell Sciences: MitoTracker® Green FM #9074


MitoTrackerDeep Red FM
MitoTrackerDeep Red FM is well retained after fixation allowing for further sample processing and immunostaining. Excitation: 644 nm, Emission: 665 nm, 
Invitrogen:  MitoTracker™ Deep Red FM M22426
Cell Sciences: MitoTracker® Deep Red FM #8778


General Protocol for Mitotracker Labelling

1.1 Preparing staining solutions. 
Dilute 1 mM MitoTracker® stock solution (see Preparing Stock Solutions for preparation) to the final working concentration in appropriate buffer or growth medium. Because the reduced forms of the MitoTracker® probes are susceptible to potential oxidases in serum, we do not recommend using complete media with these dyes. 

1.2 Staining adherent cells.
Grow cells on coverslips inside a Petri dish filled with the appropriate culture medium. When cells have reached the desired confluency, remove the media from the dish and add prewarmed (37°C) staining solution containing MitoTracker® probe.  While incubation times vary depending on the model system and probe used, incubation for 15–45 minutes under growth conditions appropriate for the particular cell type is generally sufficient but may need to be optimized. After staining is complete replace the staining solution with fresh prewarmed media or buffer and observe cells using a fluorescence microscope or fluorescence microplate reader. If the cells are to be fixed and permeabilized, continue to Fixation and Permeabilization after Staining. 

1.3 Staining suspension cells. 
Centrifuge to obtain a cell pellet and aspirate the supernatant. Resuspend the cells gently in prewarmed (37°C) staining solution containing the MitoTracker® probe (prepared in step 1.1). While incubation times vary depending on the model system and probe used, incubation for 15–45 minutes under growth conditions appropriate for the particular cell type is generally sufficient but may need to be optimized. After staining is complete, re-pellet the cells by centrifugation and resuspend cells in fresh prewarmed medium or buffer.

2.1 Washing the cells. 
After staining, wash the cells in fresh, pre-warmed buffer or growth medium.

 2.2 Fixing the cells. 
Carefully remove the medium/buffer covering the cells, and replace it with freshly prepared, pre-warmed buffer or growth medium containing 2–4% formaldehyde.
For MitoTracker® Red CMXRos, fixing with 3.7% formaldehyde in complete growth medium at 37°C for 15 minutes works well for endothelial cells.

 2.3 Rinsing the cells. 
After fixation, rinse the cells several times in a buffer

2.4 Analyze the cells using downstream methods such as microscopy, flow cytometer etc.


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