Mitochondrial Membrane Potential Detection Kit

Mitochondrial Membrane Potential detection kit provides an early indication of the initiation of cellular apoptosis. This process is typically defined as the collapse in the electrochemical gradient across the mitochondrial membrane, as measured by the change in the membrane potential. The insertion and oligomerization of proapoptotic proteins BID, BAK, BAX or BAD create pores which could dissipate the trans-membrane potential and release cytochrome C into the cytoplasm. The Mitochondrial Membrane Potential Detection Kit uses a unique cationic dye (5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolylcarbocyanine iodide) to signal the loss of the mitochondrial membrane potential. In healthy cells, the dye stains the mitochondria bright red. The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, bearing a delocalized positive charge, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, J- aggregates form which become fluorescent red. In apoptotic cells, the mitochondrial membrane potential collapses, and the dye cannot accumulate within the mitochondria. In these cells the dye remains in the cytoplasm in a green fluorescent monomeric form. Apoptotic cells, showing primarily green fluorescence, are easily differentiated from healthy cells which show red and green fluorescence. The aggregate red form has absorption/emission maxima of 585/590 nm. The green monomeric form has absorption/ emission maxima of 510/527 nm. Both apoptotic and healthy cells can be visualized simultaneously by fluorescence microscopy using a wide band-pass filter suitable for detection of fluorescein and rhodamine emission spectra. The dye reagent is easy to use. Simply dilute the reagent in cell culture medium and add to the cells. After a 15 minute incubation, wash the cells and analyze by flow cytometry or fluorescence microscopy or fluorescence plate reader.