Mitochondria Research

Science Note

[Feb. 6, 2024]                                                                                                                                                                                                                            Previous Science Note
Mitochondrial Metabolism and Oxidative Stress

Mitochondrial metabolism involves the biochemical processes within mitochondria that convert nutrients into energy and building blocks necessary for cell function, primarily through the citric acid cycle and oxidative phosphorylation. This energy production produces adenosine triphosphate (ATP), the cell's primary energy currency. Oxidative stress occurs when there's an imbalance between the production of reactive oxygen species (ROS) in the mitochondria and the cell's ability to detoxify these harmful byproducts or repair the resulting damage. Over time, excessive oxidative stress can lead to cellular damage that contributes to aging and several diseases, including neurodegenerative disorders and cancer. 

Autoregulatory control of mitochondrial glutathione homeostasis
Click here for the original article: Yuyang Liu, et. al., Science, 2023.

ApoE enhances mitochondrial metabolism via microRNA-142a/146a-regulated circuits that suppress hematopoiesis and inflammation in hyperlipidemia
Click here for the original article: Tuan Anh Phu et. al., Cell Reports, 2023.

Mitochondrial protein C15ORF48 is a stress-independent inducer of autophagy that regulates oxidative stress and autoimmunity
Click here for the original article: Yuki Takakura et. al., Nature Communications, 2024.

Point of Interest
- The mitochondrial glutathione (GSH) transporter, SLC25A39, undergoes rapid degradation by the mitochondrial protease AFG3L2.

- Depletion of GSH dissociates AFG3L2 from SLC25A39, which triggers enhancement in the uptake of GSH by mitochondria.

- This regulatory mechanism is dependent on a putative iron-sulfur cluster within SLC25A39, linking the control of mitochondrial iron homeostasis to GSH import.

Point of Interest
- Apolipoprotein E (ApoE) elevates the levels of miR-146a in myeloid cells and hematopoietic stem and progenitor cells, leading to decreased glucose absorption and glycolysis.

- By diminishing miR-142a levels, ApoE enhances fatty acid oxidation, thereby improving mitochondrial metabolism.

- ApoE plays a critical role in regulating immune cell metabolism, influencing hematopoiesis and inflammation in conditions of hyperlipidemia.

Point of Interest
- The mitochondrial protein C15ORF48 reduces mitochondrial membrane potential and intracellular ATP levels, resulting in the activation of Unc-51-like kinase 1.

- C15ORF48-dependent induction of autophagy upregulates intracellular glutathione levels and promotes cell survival by reducing oxidative stress.

- Mice deficient in C15orf48 show a reduction in stress-independent autophagy in thymic epithelial cells (TECs).

- C15orf48-/- mice develop autoimmunity, suggesting that stress-independent autophagy in TECs is critical for thymic self-tolerance.

Related Techniques
           Oxygen consumption rate assay Extracellular OCR Plate Assay Kit
           Mitochondrial superoxide detection MitoBright ROS - Mitochondrial Superoxide Detection NEW
           Mitophagy Detection Mitophagy Detection Kit and Mtphagy Dye
           Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit / MT-1 MitoMP Detection Kit
           Glycolytic/Mitochondrial activity detection Glycolysis/JC-1 MitoMP Assay Kit NEW
           Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
           Mitochondrial staining (Long-Term Visualization) MitoBright LT Green, Red, Deep Red
           Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
           Antibody/Protein labeling with fast and
           high recovery
Fluorescein, Biotin, and Peroxidase Labeling Kit - NH2
Related Applications

Inhibition of Mitochondrial Electron Transport Chain

Antimycin stimulation of Jurkat cells was used to evaluate the changes in cellular state upon inhibition of the mitochondrial electron transport chain using a variety of indicators.

The results showed that inhibition of the electron transport chain resulted in (1) a decrease in mitochondrial membrane potential and (2) a decrease in OCR. In addition, (3) the NAD+/NADH ratio of the entire glycolytic pathway decreased due to increased metabolism of pyruvate to lactate to maintain the glycolytic pathway, (4) GSH depletion due to increased reactive oxygen species (ROS), and (6) increase in the NADP+/NADPH ratio due to decreased NADH required for glutathione biosynthesis were observed. 


Mitochondrial Superoxide Detection in Senescent Cells

Background fluorescence caused by lipofuscin can be minimized by using a better fluorescent probe, as tested in TIG-1 cells. 

Lipofuscin accumulates in senescent cells, causing increased background fluorescence during observation. To minimize the effects of endogenous fluorescence from lipofuscin and other substances, a better fluorescent probe was tested in TIG-1 cells. Company T's product exhibited endogenous fluorescence, while MitoBright ROS Deep Red showed less background fluorescence. Researchers should compare sensitivity, wavelength, and channels and select the appropriate fluorescent probe to minimize endogenous fluorescence for accurate cellular senescence research.

Products in Use
  - MitoBright ROS - Mitochondrial Superoxide Detection

 


 

Selection guide for mitochondria-related reagents

Mitochondria research is very multi-faceted, because the multi-functional organelle is not only involved in energy production in a cell, but other additional cellular functions. The active cycle of mitochondrial fusion and division induces morphological changes, which is called mitochondrial dynamics. Abnormalities in morphological control of mitochondria are associated with neurodegenerative diseases, metabolic disorders, aging, and so on. Therefore, the demand for long-term observation of mitochondrial dynamics has recently been increasing.

Selection Guide of Reagents

The following table lists reagents for mitochondrial research designed to stain and detect mitochondria (MitoBright LT, MitoTracker, etc.), mitochondrial membrane potential (JC-1, TMRM, TMRE, etc.), reactive oxygen species AKA ‘ROS’ (MitoBright ROS, MitoSOX, etc.), mitophagy, and lipid peroxides.

Mitophagy

Mitophagy
Reagent Mtphagy Dye Keima-Red
Principle Mtphagy Dye (included in Mitophagy Detection Kit) is a pH-sensitive fluorescent probe that accumulates in mitochondria and emits red fluorescence due to acidic conditions in a lysosome. A pH-sentitive ratiometric fluorescent protein. The excitation spectrum changes accoring to pH. This protein shows high fluorescence ratio (Ex. 550 nm/440 nm) values in a lysozome.
Fixed cell staining
Live-cell staining Yes Yes
Fixation after live-cell staining
Staining time > 30 min
Ex / Em 530 / 700 440, 550 / 620
Product code MD01MT02

Lipophilic peroxide / Singlet oxygen / Superoxide

  Lipophilic peroxide Singlet oxygen Superoxide Superoxide
Reagent MitoPeDPP Si-DMA MitoBright ROS Deep Red MitoSOX
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with lipophilic peroxides in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with singlet oxigen generated in mitochondria to emit red fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and reacts with superoxide generated in mitochondria to emit red fluorescence.
Fixed cell staining
Live-cell staining Yes Yes Yes Yes
Fixation after live-cell staining
Staining time > 15 min > 45 min > 10 min > 10 min
Ex / Em 452 / 470 644 / 670 540 / 670 510 / 590
Product code M466 MT05 MT16

Membrane potential

Membrane potential
Reagent JC-1 MT-1 TMRM, TMRE
Principle A cell-permeant ratiometric fluorescent dye that accumulates in intact mitochondria due to the membrane potential. The excitation spectrum changes according to the mitochondria membrane potential. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. MT-1 is extremely photostable and more sensitive than JC-1 and can provide equivalent detection sensitivity to TMRE. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Diffusion of the probes occurs in a damaged mitochondria that has the decreased membrane potential.
Fixed cell staining
Live-cell staining Yes Yes Yes
Fixation after live-cell staining Yes
Staining time 30-60 min 30 min 30-60 min
Ex / Em Monomer: 514 / 529
J-aggregation: 585/590
530-560 / 570-640 550 / 575
Product code MT09 MT13

Mitochondria staining

Mitochondria staining
Reagent MitoBright LT series MitoBright IM Red MitoTracker series Rhodamine 123
Principle Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential and covalently binds to proteins and other biomolecules. Cell-permeant fluorescent dyes that accumulate in intact mitochondria due to the membrane potential. Cell-permeant fluorescent dye that accumulates in intact mitochondria due to the membrane potential.
Fixed cell staining
Live-cell staining Yes Yes Yes Yes
Fixation after live-cell staining Yes Yes
Staining time 30 min 30 min 15-45 min > 15 min
Ex / Em 493 / 508, 547 / 563, 643 / 663 548 / 566 490 / 516 ~
644 / 665
507 / 529
Product code MT10, MT11,MT12 MT15 R233

Metal Ion Detection

   Iron ion (Fe2+) Calcium ion (Ca2+)
Reagent Mito-FerroGreen Rhod2-AM
Principle A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with ferrous ion in mitochondria to emit green fluorescence. A cell-permeant fluorescent probe that accumulates in mitochondria and specifically reacts with calcium ion in mitochondria to emit red fluorescence.
Fixed cell staining
Live-cell staining Yes Yes
Fixation after live-cell staining
Staining time 30 min 30-60 min
Ex / Em 505 / 535 553 / 576
Product code M489 R002 
Application Products
Mitophagy Detection Mitophagy Detection Kit
Mitochondrial Phospholipid Peroxidase Detection MitoPeDPP
Mitochondrial Ferrous Ion Detection Mito-FerroGreen
Mitochondrial Superoxide MitoBright ROS - Mitochondrial Superoxide Detection
Mitochondrial Singlet Oxygen Detection Si-DMA for Mitochondrial Singlet Oxygen Imaging
Mitochondrial Membrane Potential JC-1 MitoMP Detection Kit
MT-1 MitoMP Detection Kit
Mitochondria Staining MitoBright LT Green
MitoBright LT Red
MitoBright LT Deep Red
MitoBright IM Red for Immunostaining

Product Classification

Product Classification