MitoView™ Mitochondrial Transition Pore Assay for Imaging

The mitochondrion plays a vital role in the processes of apoptotic and necrotic cell death. The mitochondrial permeability transition pore is a nonspecific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death. The MitoView™ Mitochondrial Transition Pore Assay is based on published experimentation for mitochondrial transition pore opening. This assay employs calcein AM, a colorless and nonfluorescent esterase substrate, and CoCl₂ , a quencher of calcein fluorescence, to selectively label mitochondria.

The MitoView™ Mitochondrial Transition Pore Assay provides a direct method of measuring mitochondrial permeability transition pore opening. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, intracellular esterases cleave the acetoxymethyl esters to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes. The fluorescence from cytosolic calcein is quenched by the addition of CoCl₂ , while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl₂ can also be treated with an ionophore, ionomycin, to allow entry of excess Ca²⁺ into the cells to trigger mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence.