Mitochondria Research

Science Note

[Dec. 3, 2024]                                                                                                                                                                                                                            Previous Science Note
Boosting T-cells with Mitochondrial Activation to Treat Tumours

Recent research shows that mitochondria are critical for T cell function, regulating metabolic pathways essential for immune responses. Here are some of the papers that show mechanisms and strategies, such as mitochondrial transfer or genetic modification, to improve mitochondrial health and reinvigorate T cell activity against tumours.

Mitochondria play a critical role in T cell function, providing energy and regulating metabolic pathways essential for immune responses. In cancer, the tumour microenvironment induces mitochondrial dysfunction in T cells, leading to exhaustion and reduced anti-tumour efficacy. Strategies to improve mitochondrial health, such as mitochondrial transfer or genetic modification, have shown promise in reinvigorating T cell activity against tumours. Targeting mitochondrial pathways in T cells offers a novel approach to improving cancer immunotherapies and overcoming tumour-induced immunosuppression.

Intercellular nanotube-mediated mitochondrial transfer enhances T cell metabolic fitness and antitumor efficacy
Click here for the original article: Jeremy G. Baldwin, et. al., Cell, 2024.

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function
Click here for the original article: Matteo Morotti, et. al., Nature, 2024.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy
Click here for the original article: Jack D. Chan, et. al., Nature, 2024.

Point of Interest

- Bone marrow stromal cells transfer mitochondria to CD8+ T cells via nanotubes, requiring Talin 2 for optimal transfer.

- Mitochondria-boosted T cells exhibit improved respiration, tumor infiltration, and reduced exhaustion, enhancing antitumor responses.

- Intercellular mitochondrial transfer offers a new organelle-based therapy, advancing next-generation cell therapies for cancer treatment.

Point of Interest

- Prostaglandin E2 (PGE2) impairs IL-2 sensing in CD8+ tumour-infiltrating lymphocytes (TILs) by downregulating IL-2 receptor components, causing oxidative stress and cell death.

- Blocking PGE2 signalling during TIL expansion restores IL-2 responsiveness, increasing TIL proliferation and improving tumour control in adoptive cell therapy.

- Targeting PGE2 pathways enhances IL-2-driven T cell expansion, offering new strategies to improve cancer immunotherapy outcomes.

Point of Interest

- CAR T-cell therapy is less effective in solid tumours due to the immunosuppressive microenvironment that causes T-cell dysfunction.

- Overexpression of FOXO1 enhances the stem-like phenotype, mitochondrial fitness and persistence of CAR T cells, thereby improving antitumour efficacy.

- Engineering FOXO1 in CAR T cells offers a promising strategy to increase efficacy against solid tumours in cancer therapy.
Related Techniques
Mitochondrial membrane potential detection JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Mitochondrial superoxide detection MitoBright ROS Deep Red - Mitochondrial Superoxide Detection
Mitophagy or autophagy detection Mitophagy Detection Kit, Autophagic Flux Assay Kit
Mitochondrial Staining MitoBright LT Green / Red / Deep Red
Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit, Extracellular OCR Plate Assay Kit
Lipid Droplet Staining Lipi-Blue/ Green/ Red/ Deep Red
Intracellular / mitochondrial ferrous ion (Fe2+) detection FerroOrange, Mito-FerroGreen
Intracellular / mitochondrial lipid peroxidation detection Liperfluo, MitoPeDPP
Apoptosis detection in multiple samples NEW Annexin V Apoptosis Plate Assay Kit
Total ROS detection Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Cell Proliferation / Cytotoxicity Assay Cell Counting Kit-8, Cytotoxicity LDH Assay Kit-WST
Related Applications

Simultaneously evaluation of mitochondrial superoxide and membrane potential

After HeLa cells were washed with HBSS, co-stained with MitoBright ROS Deep Red and mitochondrial membrane potential staining dye (JC-1: code MT09), and the generated mitochondrial ROS and membrane potential were observed simultaneously. As a result, the decrease in mitochondrial membrane potential and the generation of mitochondrial ROS are simultaneously observed.

＜Imaging Conditions＞(Confocal microscopy)
JC-1: Green Ex = 488, Em = 490-520 nm, Red: Ex = 561, Em = 560-600 nm
MitoBright ROS ：Ex = 633 nm, Em = 640-700 nm
Scale bar: 10 μm

＜Examination Conditions＞(Plate Reader)Tecan, Infinite M200 Pro
JC-1: Green Ex=480-490 nm, Em=525-545 nm; Red: Ex= 530-540 nm, Em=585-605 nm
MitoBright ROS: Ex=545-555 nm, Em = 665-685 nm

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. 

 
 
 
 
 

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 MD01, MT02 –

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

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