Mitochondria play a central role in the biochemical processes associated with the life and death stages of eukaryotic cells. Under normal physiological conditions, a membrane-based proton pump generates an electrochemical gradient, enabling the production of ATP to drive cellular energy dependent processes. The oxidation of glucose and fatty acids by enzymes associated with the mitochondrial respiratory chain establishes a proton and pH gradient across the mitochondrial inner membrane, resulting in a transmembrane electrical potential gradient (ΔΨm) of -80 to -120 mV and a pH gradient of 0.5-1.0 pH units.
Depolarization of the inner mitochondrial membrane can lead to an opening of the mitochondrial permeability transition pore (PTP). This results in the leakage of intermembrane proteins, including cytochrome C, that facilitate the induction of apoptosis through apoptosome formation. Caspase activation has been shown to accelerate the process of ΔΨm loss. Moreover, a feedback mechanism that results in the generation of reactive oxygen species (ROS) further accelerates the rate of cell death. Because mitochondrial dysfunction has been closely tied to such neurodegenerative diseases as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, mitochondria remain an important organelle of study.
Loss of mitochondrial ΔΨm, indicative of apoptosis, can easily be detected using lipophilic, cationic fluorescent redistribution dyes such as ICT’s Fluorescent Mitochondrial Membrane Depolarization Assay reagents: tetramethylrhodamine ethyl ester (TMRE), tetramethylrhodamine methyl ester (TMRM), and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide (JC-1). These dyes have a delocalized positive charge dispersed throughout their molecular structure, and yet their lipophilic solubility enables them to be readily membrane permeant and penetrate living cells. They redistribute across cell membranes according to the Nernst equation in a voltage-dependent manner. Accordingly, they possess a low membrane partition coefficient: a low tendency to non-specifically associate with intracellular organelles and macromolecules. These excellent potentiometric dyes also exhibit minimal self-quenching, low cytotoxicity, and are reasonably photostable. The Fluorescent Mitochondrial Membrane Depolarization Assay dyes exhibit very low toxicity and display rapid and reversible membrane equilibration properties.
ICT’s TMRE and TMRM assay kits easily distinguish between healthy, non-apoptotic cell populations and cell populations that are transitioning into an apoptotic state. Inside a healthy, non-apoptotic cell, the lipophilic TMRE or TMRM dye, bearing a delocalized positive charge, enters the negatively charged mitochondria where it accumulates and fluoresces orange upon excitation. When the mitochondrial ΔΨm collapses in apoptotic cells, TMRE or TMRM no longer accumulates inside the mitochondria, instead becoming more evenly distributed throughout the cytosol. When dispersed in this manner, overall cellular fluorescence levels drop dramatically. Healthy cells fluoresce orange, whereas cells with depolarized mitochondria exhibit lower levels of orange fluorescence.
TMRE and TMRM kits can be used in conjunction with existing research protocols. Grow cells following the usual cell cultivation protocol. If using an apoptosis induction model system, induce apoptosis according to the existing procedure, reserving a non- induced population of cells as a control. Once apoptosis has been induced or the mitochondrial membrane has been depolarized by a known method, such as using CCCP (included), spike Fluorescent Mitochondrial Membrane Depolarization Assay dye solution into each sample and control. Incubate the cells for 15-30 minutes at 37°C to allow Fluorescent Mitochondrial Membrane Depolarization Assay reagent to equilibrate within the polarized mitochondria. If cells are not undergoing some form of metabolic or apoptotic stress, the mitochondrial ΔΨm will remain intact, and Fluorescent Mitochondrial Membrane Depolarization Assay reagent will accumulate within the slightly negative/alkaline environment of the mitochondria and fluoresce brightly upon excitation. If the cells are apoptotic, the mitochondrial ΔΨm will break down, causing Fluorescent Mitochondrial Membrane Depolarization Assay reagent to disperse throughout the cell cytosol. This results in a dramatic reduction in the fluorescence of the affected mitochondria, and as a result, overall cellular fluorescence is diminished significantly.
Fluorescent Mitochondrial Membrane Depolarization Assays are for research use only. Not for use in diagnostic procedures.
- Prepare samples.
- Create controls with CCCP.
- Dilute 10X Assay Buffer 1:10 with diH2O.
- Reconstitute MitoPT TMRM with DMSO.
- Dilute MitoPT TMRM with 1X Assay Buffer.
- Add MitoPT TMRM to each sample.
- Incubate 15-30 minutes.
- Wash cells: add 1X Assay Buffer and spin cells.
- Remove supernatant and resuspend cells for analysis.
- Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. MitoPT TMRM excites at 548 nm and emits at 573 nm (orange).
Product Specific References
|Blanco, D., et al. 2022. PTEN directs developmental and metabolic signaling for innate-like T cell fate and tissue homeostasis. Nature Cell Biology, 1642-1654.
|Lim, S.A., et al. 2021. Lipid signalling enforces functional specialization of Treg cells in tumours. Nature.
|Huang, H., et al. 2021. In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions. Cell.
|Cai, H., et al. 2021. Critical Role of Lama4 for Hematopoiesis Regeneration and Acute Myeloid Leukemia Progression. Blood.