Is MitoTracker green fixable?
Most of the MitoTracker® dyes are well-retained following fixation and permeabilization using the protocol described below. However, MitoTracker® Green FM and MitoTracker® Red FM are not retained well after fixation. 2.1 Washing the cells.
How do you dissolve a MitoTracker?
Dissolve one vial (50 µg) of MitoTracker Green FM (Invitrogen M7514) in DMSO to prepare a stock solution of 1 mm. Cover with aluminum foil to minimize light exposure. Store at or below −20°C.
How does MitoTracker orange work?
MitoTracker® Orange CMTMRos is an orange-fluorescent dye that stains mitochondria in live cells and its accumulation is dependent upon membrane potential. The dye is well-retained after aldehyde fixation.
How do you use a MitoTracker?
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- Remove medium from the cells and add prewarmed medium with MitoTracker (most commonly used concentration is in the range of 300-400 nmol).
- Incubate for 45 to 60 minutes.
- Exchange the medium with regular one and incubate for 5 min, repeat 2 more times.
How does MitoTracker deep red work?
MitoTracker® Deep Red binds thiol‐reactive chloromethyl groups in the mitochondrial membrane and is retained after cell fixation, whereas MitoTracker® Green binds free thiol groups of cysteine residues belonging to mitochondrial proteins and can only be used with non‐fixed cells.
Can you use MitoTracker on fixed cells?
The mitochondria can be labelled with mitotracker because this reagent present electronic mitochondrial potential affinity. If you fix the cells before use mitotracker will not work because you don’t have electronic mitochondrial potential. If you have already the fixed cells, you need use antibody.
How do you make a MitoTracker?
- Grow bloodstream form T.
- Prepare a 1 mM stock of Mitotracker (Invitrogen/Molecular Probes, #M7512) in DMSO, i.e. 50 μg CMXROS in 94 μl.
- Add 1 μl of the stock Mitotracker solution to a 10ml culture, giving a final concentration of 100 nM, and incubate at 37oC for 5 minutes.
What does MitoTracker measure?
MitoTracker green (MTG) and nonylacridine orange (NAO) are fluorescent probes which have been commonly used to assess mitochondrial mass. This is based on the assumption that both MTG and NAO accumulate in mitochondria regardless of the mitochondrial transmembrane potential (ΔΨm).
Is MitoTracker deep red dependent on membrane potential?
Our data and previous reports suggest that MitoTracker Deep Red is a mitochondrial potential-dependent dye and therefore should not be used to quantitatively assess mitophagy.
What is a MitoTracker?
MitoTracker is a commercially available fluorescent dye (Invitrogen/Molecular Probes) that, like the aforementioned dyes, labels mitochondria within live cells utilizing the mitochondrial membrane potential. In addition, it can be used in experiments in which multiple labeling diminishes mitochondrial function.