Mitopt Jc-1 Assay Kit

Background/Introduction: Mitochondria play a central role in the biochemical processes associated with the life and death stages of eukaryotic cells1. Under normal physiological conditions, a membrane-based proton pump generates an electrochemical gradient, enabling the production of ATP to drive cellular energy-dependent processes2. 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 (delta?m) of -80 to -120 mV and a pH gradient of 0.5-1.0 pH units3, 4. Depolarization of the inner mitochondrial membrane can lead to an opening of the mitochondrial permeability transition pore (PTP)5. This results in the leakage of intermembrane proteins, including cytochrome c, that facilitate the induction of apoptosis through apoptosome formation6. Caspase activation has been shown to accelerate the process of delta?m loss7. Moreover, a feedback mechanism that results in the generation of reactive oxygen species further accelerates the rate of cell death7. 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 study8. Loss of mitochondrial delta?m, indicative of apoptosis, can easily be detected using lipophilic, cationic fluorescent redistribution dyes such as MitoPT® reagents: tetramethylrhodamine ethyl ester (TMRE), tetramethylrhodamine methyl ester (TMRM), and 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1)9. 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 cells9-11. They redistribute across cell membranes according to the Nernst equation in a voltage-dependent manner9-11. Accordingly, they possess a low membrane partition coeff icient; meaning 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 photostable11. The MitoPT dyes exhibit very low toxicity and display rapid and reversible membrane equilibration properties11. MitoPT JC-1 assay kits easily distinguish between healthy, non-apoptotic cell populations and those cell populations that are transitioning into an apoptotic state. Inside a healthy, non-apoptotic cell, the lipophilic JC-1 dye, bearing a delocalized positive charge, enters the negatively charged mitochondria where it accumulates10. When the mitochondrial delta?m collapses in apoptotic cells, MitoPT JC-1 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 and green, whereas cells with depolarized mitochondria fluoresce green and exhibit lower levels of orange fluorescence. MitoPT JC-1 kits can be used in conjunction with other existing research protocols. Grow cells following the usual cell cultivation protocol. If using an apoptosis induction model system, simply induce apoptosis according to the existing procedure, reserving a non-induced population of cells as a control. Once apoptosis has been induced in the cells, or the mitochondrial membrane has been depolarized by another method, such as using CCCP (included in the kit), spike MitoPT JC-1 dye solution into each sample and control. Incubate the cells for 15-30 minutes at 37 degree C to allow MitoPT JC-1 to equilibrate within the polarized mitochondria. If the cells are not undergoing some form of metabolic or apoptotic stress, the mitochondrial delta?m will remain intact, and MitoPT JC-1 will accumulate within the slightly negative/alkaline environment of the mitochondria and fluoresce brightly upon excitation. If the cells are apoptotic, the mitochondrial delta?m will break down, causing MitoPT JC-1 to disperse throughout the cell cytosol. This results in a dramatic reduction in the fluorescence of the aff ected mitochondria, and as a result, overall cellular fluorescence is diminished significantly. MitoPT® is for research use only.

Small volumes of MitoPT JC-1 assay kit vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container`s cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

MBS258016 is an assay kit targeting MitoPT JC-1. Also known as MitoPT JC-1 Assay Kit (100 tests).