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).

What does MitoTracker green bind to?

Mitotracker Green is commonly used as a marker for mitochondrial mass and it binds to mitochondrial proteins (free thiol groups of cystein residues).

What does MitoTracker deep red measure?

Description. MitoTracker® Deep Red FM is a far red-fluorescent dye (abs/em ∼644/665 nm) that stains mitochondria in live cells and can be used for mitochondrial localization. The dye is well-retained after aldehyde fixation and even subsequent permeabilization with detergents.

How do you use a MitoTracker?

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1. Remove medium from the cells and add prewarmed medium with MitoTracker (most commonly used concentration is in the range of 300-400 nmol).
2. Incubate for 45 to 60 minutes.
3. Exchange the medium with regular one and incubate for 5 min, repeat 2 more times.

What is MitoTimer?

MitoTimer is a novel fluorescent reporter protein that can reveal new insights into mitochondrial dynamics within cells. Coupled with organelle flow cytometry, it offers new opportunities to investigate mitochondrial subpopulations by biochemical or proteomic methods.

How do you measure mitochondrial function?

Traditionally, mitochondrial function has been assessed with Clark-type electrode probes for measuring oxygen consumption, luminescent ATP assays for quantification of total energy metabolism, and MTT or Alamar Blue for determination of metabolic activity.

Are mitochondria green?

Viewed through a fluorescein longpass optical filter, fluorescence of the mitochondria of cells stained by rhodamine 123 appears yellow-green.

Why do we stain the mitochondria?

Many of the mitochondria dyes are dependent on mitochondrial membrane potential and can therefore be used to monitor membrane potential changes that occur during processes such as apoptosis. MitoView™ Dyes stain the mitochondria in living cells.

What is the half life of mitochondria?

Studies of cardiac mitochondria revealed an average half-life of 17 days under steady state conditions [115].

How do you measure Mitophagy?

Mitophagy can be quantified by fluorescence imaging using a mitochondria-matrix targeted Keima (mt-Keima) fluorescent protein in cells and in vivo. Similar to MitoTimer, Keima is targeted to the mitochondrial matrix via fusion with COX8 to make mt-Keima.

How do you study mitochondrial dysfunction?

A very common approach to address mitochondrial bioenergetic dysfunction is to measure the expression, concentration or maximum activity of a few candidate electron transport complexes or metabolic enzymes, such as complex I, complex IV or tricarboxylic acid-cycle enzymes.

How do you check for mitochondrial damage?

They include: biochemical tests on urine, blood and spinal fluid. a muscle biopsy to examine the mitochondria and test enzyme levels. magnetic resonance imaging (MRI) of the brain and spine….Testing depends on symptoms, and may include:

1. echocardiogram.
2. electrocardiogram (EKG)
3. eye examinations.
4. hearing tests.

How is MtG used to label mitochondrial proteins?

Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE–LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species.

What are the metabolites in the mitochondrial matrix?

Additional metabolites present in the matrix are CO 2, H 2 O, O 2, ATP, ADP, and P i. Enzymes from processes that take place in the matrix. The citric acid cycle is facilitated by pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinyl-CoA synthetase, fumarase, and malate dehydrogenase.

What is the water content of the mitochondrial matrix?

The cytosolic, intermembrane space, compartment has a water content of 3.8 μl/mg protein, while the mitochondrial matrix 0.8 μl/mg protein.

What kind of markers are found in mitochondria?

Mitochondria were sub fractionated into outer membrane (OM), inter membrane space (IMS), inner membrane (IM) and mitochondrial matrix (MM). The purity of the fractions was analyzed with marker proteins including TOM20 for OM, AIF for IMS, COX4 (NB110-39115) for IM, and PDK4 for MM specificity.