In the intrinsic apoptosis pathway, mitochondrial permeability transition is an important event wherein the electrochemical gradient (referred to as delta-psi or ΔΨm) across the mitochondrial membrane collapses. This collapse occurs through the formation of channels or pores in the outer mitochondrial membrane, which involves Bax insertion and oligomerization, followed by the release of Cytochrome C into the cytoplasm.
The DePsipher™ Kit uses a unique cationic dye (5,5’6,6’-tetrachloro-1,1’,3, 3’-tetraethylbenzimidazolyl- carbocyanine iodide) to indicate the loss of ΔΨm. The dye readily enters cells and fluoresces brightly red in its multimeric form within healthy mitochondria. In apoptotic cells, the mitochondrial membrane potential collapses, and the DePsipher™ reagent cannot accumulate within the mitochondria. In these cells, DePsipher™ returns to its green fluorescent monomeric form. Apoptotic cells, showing primarily green fluorescence, are thus easily differentiated from healthy cells which show red fluorescence. The aggregate red form has absorption/emission maxima of 585/590 nm, and the green monomeric form has absorption/emission maxima of 510/527 nm. Both apoptotic and healthy cells can be visualized simultaneously by epifluorescence microscopy using a wide band-pass filter.
The DePsipher™ reagent is easy to use. Simply resuspend the reagent in reaction buffer or culture media (with or without the stabilizer solution), add to your cells, incubate for 15 to 20 minutes, wash and analyze by flow cytometry or microscopy. Visualization by microscopy allows a rapid inspection and qualification of apoptosis. Flow cytometric analysis allows easy quantitation of cell death as evidenced by mitochondrial potential breakdown.
- Simple. Just add DePsipher™ reagent to media or reaction buffer.
- Unique Stabilizer Solution improves results.
- Fast. Takes only 20 minutes.
- Flexible. View cells by epifluorescence or confocal microscopy, or analyze cells by flow cytometry.
- Flow cytometry
- Epifluorescence microscopy
- Confocal microscopy