MitoPT™ Kits
Detect changes in mitochondrial membrane permeability
ICT offers 3 assay kits that utilize JC-1, TMRE, or TMRM fluorescent potentiometric dyes for assessing mitochondrial depolarization via flow cytometry, fluorescence microscopy, and fluorescence plate reader assay formats. These unique depolarization assay kits can facilitate the simple detection of apoptosis induction or other metabolic or oxidative stress conditions in your experimental model system.
Healthy cells containing mitochondria with normal membrane proton gradient potentials will fluoresce orange/red. Apoptotic and metabolically stressed cells will lose most of their fluorescence or, if using the MitoPT JC-1 assay format, will fluoresce green.
Assay Features
Simple: Kit formats are easy to use. Just add the cell-permeant reagents to the cell culture samples and incubate; no membrane permeabilization steps are necessary. Healthy cells will fluoresce orange-red.
Fast: These assay kits provide an assessment of the mitochondrial functionality status in less than 30 minutes. The typical dye incubation protocol requires only 15-20 minutes.
Accurate: Orange-red signal is reduced in mitochondrial compromised cells, providing a clear distinction between cells with polarized mitochondria and cells with depolarized mitochondria.
Quantitative: All three MitoPT assay formats are compatible with flow cytometry, fluorescence plate reader, or fluorescence microscopy analysis methods.
Assay Principle
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 for use in driving the cells' energy dependent processes. Depolarization of the inner mitochondrial membrane potential usually leads to an opening of the mitochondrial permeability transition pore. This process allows for the release 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 loss. Because mitochondrial dysfunction has been closely tied to such neurodegenerative disease as Alzheimers, Parkinsons, and Amyotrophic Lateral Sclerosis to name a few, the assessment of mitochondrial function status remains as an important area of study.
Loss of mitochondrial membrane potential, indicative of apoptosis, can easily be detected using lipophilic, cationic, fluorescent redistribution dyes such as tetramethylrhodamine ethyl (TMRE) and methyl (TMRM) esters and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). These potentiometric dyes have a delocalized positive charge dispersed throughout their molecular structure and yet their lipophilic solubility allows them to be readily membrane permeant and penetrate living cells. The dyes redistribute across cell membranes according to the Nernst equation in a voltage dependent manner. Accordingly, they possess a low membrane partition coefficient; meaning a low tendency to non-specifically associate with intra-cellular organelles and macromolecules. These excellent potentiometric dyes also exhibit minimal self quenching, low cytotoxicity, and are reasonably photostable.
Order MitoPT Assay Kits Online
More Information:
5 Jurkat cells stained with MitoPT JC1. Healthy cells fluoresce red (2 cells on left); dying cells fluoresce green (3 cells on right).
Kit Contains:
- MitoPT reagent
- CCCP
- Assay buffer
Sample protocol:
- Culture cells up to 1 x 10^6 cells/mL.
- Induce apoptosis following your protocol.
- Create controls using CCCP or other agent.
- Reconstitute MitoPT with DMSO to form the stock concentrate (can be frozen for future use).
- Dilute stock concentrate with 1X Assay Buffer to form the working solution.
- Add the working solution directly to your cell culture for labeling.
- Incubate 15 minutes -1 hour.
- Wash cells twice.
- Analyze data using a fluorescence microscope, plate reader, or flow cytometer.