How do you measure mitochondrial membrane potential?

How do you measure mitochondrial membrane potential?

Mitochondrial function, a key indicator of cell health, can be assessed by monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in HepG2 cells.

How does valinomycin affect mitochondrial function?

Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death.

What does TMRE measure?

TMRE emits a red fluorescence that can be detected by flow cytometry or fluorescence microscopy and the level of TMRE fluorescence in stained cells can be used to determine whether mitochondria in a cell have high or low ΔΨm.

How is mitochondrial damage measured?

Measurement of the oxygen consumption rate (OCR) Oxygen consumption is an excellent read-out for mitochondrial respiratory activity. Measurement of the oxygen consumption rate (OCR) is the current experiment of choice to determine underlying mitochondrial dysfunction [36, 37].

How do we measure membrane potential?

The membrane potential is measured using a reference electrode placed in the extracellular solution and a recording electrode placed in the cell soma. The membrane potential is the difference in voltage between these two regions.

What is valinomycin used for?

Valinomycin is a potent antibiotic. It is commonly used as an agent to induce apoptosis and can also be used to calibrate potentiometric responses in membrane experiments involving potential-sensitive dyes such as di-4-ANEPPS.

How does valinomycin transport ions across the cell membrane?

Because valinomycin is uncharged and contains no ionizable groups, it acquires the charge of the complexed ion. Both the uncomplexed and complexed forms of valinomycin are able to diffuse across the membrane. Therefore, a catalytic amount of ionophore can induce the bulk transport of cations.

What does TMRE stand for?

Acronym. Definition. TMRE. tetramethyl rhodamine ethyl ester. Copyright 1988-2018, All rights reserved.

How do you measure the size of a mitochondria?

The mitochondrial length is calculated by selecting the “perimeter” parameter and dividing the corresponding values by two.

What is the mechanism of action of valinomycin on mitochondria?

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM.

What is the K+ diffusion potential of valinomycin?

At high concentrations of valinomycin the plasma membrane becomes permeable to Rb+ and both TPMP+ and Rb+ distribution give a measure of the K+ diffusion potential. Valinomycin Mitochondria Plasma membrane 1. INTRODUCTION Valinomycin is a cyclic peptide antibiotic which catalyses K+ transport across membranes [1].

What is valinomycin ionophore?

Valinomycin ( Figure 2.2) is a mobile carrier ionophore that catalyses the electrical uniport of Cs +, Rb +, K +, or NH 4 +. The ability to transport Na + is at least 10 4 less than for K +.

What is the molecular geometry of valinomycin?

The circular structure is a macrocyclic molecule with the 12 carbonyl oxygens facing the inside of the ring where they chelate a single K +. The outside surface of valinomycin is coated with nine hydrophobic side chains of d – and l -valine and l -hydroxyvaleric acid. The polar interior of valinomycin precisely fits one K +.