MitoPT™ Kit for Detection of Apoptosis through Mitochondrial Functionality

An early indication of apoptosis involves a collapse in the electrochemical gradient across the mitochondrial membrane. This is mediated by the opening of permeability transition (PT) pores and is known as the mitochondrial PT event. It is measured by the change in the mitochondrial membrane potential.

Changes in the mitochondrial Dy lead to the insertion of pro-apoptotic proteins into the membrane and possible oligomerization of BID, BAK, BAX or BAD. This may result in pores in the mitochondrial membrane, which dissipate the transmembrane potential, thereby releasing cytochrome c into the cytoplasm. Loss of mitochondrial Dy can be detected by a unique fluorescent cationic dye, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzamidazolocarbocyanin iodide, also known as JC-1. This dye is lipophilic and has a delocalized positive charge, which enables it to enter the negatively-charged healthy mitochondria. JC-1 has been incorporated into the MitoPT™ Kit, available from AbD Serotec, for the simple and reproducible detection of the mitochondrial PT event in apoptotic cells.

The MitoPT™ reagent easily penetrates cells and enters the mitochondria inside. If the cell is not undergoing apoptosis, the mitochondrial Dy will be intact and the MitoPT™ reagent aggregates (known as J-aggregates) in the mitochondria and fluoresces red. In apoptotic cells, when the mitochondrial Dy collapses, the MitoPT™ reagent no longer accumulates inside the mitochondria and diffuses throughout the cell. Once dispersed, the reagent assumes a monomeric form and fluoresces green. Thus, cells in the process of apoptosis will contain less red J-aggregates in the mitochondria, and the entire cell will appear more green, as the mitochondrial Dy disintegrates, thereby dispersing the MitoPT™ dye (see Figure). This allows an easy distinction between non-apoptotic red fluorescent cells and apoptotic green fluorescent cells which can be read with a flow cytometer, fluorescence microscope or fluorescence plate reader.

These kits are part of our large range of high quality antibodies and detection kits for studying apoptosis. MitoPT™ is a trademark of Immunochemistry Technologies Inc. USA.

Further Reading
Antonsson, B., et al. (2000) Biochem J. 345:271-278
Cossarizza, A., et al. (1993) Biochem. Biophys. Res. Commun. 197:40-45
Desagher, S., et al. (1999) J. Cell Biol. 144:891-901
Dunn, C. et al. (2007) J. Exp. Med. 204:667-680
Jelley, E. E. (1993) Nature 139:631-632
Luo, X., et al. (1998) Cell 94:481-490
Narita, M., et al. (1998) Proc. Natl. Acad. Sci. 95:14681-14686
Reers, M., et al. (1991) Biochemistry 30:4480-4486
Smiley, S. T., et al. (1991) Proc. Natl. Acad. Sci. 88:3671-3675