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The MitoLight™ Apoptosis Detection Kit utilizes a
lipophilic cation, termed as MitoLight™, as a mitochondrial activity
marker. MitoLight™ is a mitochondrial dye that stains mitochondria in
living cells in a membrane potential-dependent fashion. The monomer is
in equilibrium with so-called J-aggregates, which are favored at
higher dye concentration or higher mitochondrial membrane potential.
MitoLight™ partitions differently in healthy cells than in apoptotic
cells. Therefore, it has been possible to use a fluorescence ratio-ing
technique to study mitochondrial membrane potentials. In healthy
cells, the dye accumulates and aggregates in the mitochondria, giving
off a bright red fluorescence (l em = 585-590 nm). In apoptotic cells
with altered mitochondrial membrane potential, the dye in its
monomeric form stays in the cytoplasm, fluorescing green (l em =
527-530 nm), providing a ready discrimination between apoptotic and
nonapoptotic cells. The fluorescence can be observed by fluorescence
microscopy using a band-pass filter (detects FITC and rhodamine) or
analyzed by flow cytometry using FITC channel for green monomers (Ex/Em
= 488/530) and PI channel for red aggregates (Ex/Em = 488/585).
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