Mitochondria-Related Events

Caspases are cysteinyl proteases that are synthesized as inactive cytosolic precursor molecules that become converted by proteolytic cleavage to their active form during apoptosis. Active caspases cleave a variety of factors including other caspases, contributing to their activation or inactivation. There are 14 known caspases (1-14) with caspase 3, caspase 8 and caspase 9 playing pivotal roles in apoptosis.

The interactions between different caspases are very complex and depend on cell type and induction method. As an example, in the Fas dependent apoptosis pathway, caspase 8 cleaves procaspase 3 into its active caspase 3 form which, in turn, cleaves a variety of substrates including poly (ADP-ribose) polymerase (PARP). The amino acid sequence targeted by caspase 3 is DEVD (Asp- Glu- Val- Asp) and was used to design the caspase irreversible inhibitor Z-DEVD-FMK.

Caspase inhibitors inactivate specific caspases allowing study of their impact on pathways of interest. The caspase inhibitors are synthetic peptides coupled to fluoromethylketone (FMK) which irreversibly binds the caspase. The inhibitors contain a methyl ester amino acid (O-Me) to enhance membrane permeability.

Among the events taking place during apoptosis are cytochrome c release into the cytoplasm, caspase activation, change of mitochondrial potential, phosphatidylserine flipping, and DNA fragmentation. All these phenomena can be studied in conjunction with these caspase inhibitors.

These Apoptosis Detection Kits use a novel approach to detect active caspases: using caspase inhibitors coupled to a fluorochrome. Once inside the cell, the labeled inhibitor binds covalently to the active caspase. These inhibitors are cell permeable and non-cytotoxic. For kits using green fluorescence, a carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspases is used.

Cells that contain the bound inhibitor can be analyzed by 96-well-plate based fluorometry for quantitation fluorescence microscopy for qualitative analysis, or flow cytometry for quantitation.
 

These Apoptosis Detection Kits use a novel approach to detect active caspases: using caspase inhibitors coupled to a fluorochrome. Once inside the cell, the labeled inhibitor binds covalently to the active caspase. These inhibitors are cell permeable and non-cytotoxic. For kits using red fluorescence, a sulforhodamine-labeled fluoromethyl ketone peptide inhibitor of caspases is used. Trevigen, Inc. also offers a line of apoptosis detection kits that use green carboxyfluorescein-labeled inhibitors.

Cells that contain the bound inhibitor can be analyzed by 96-well-plate based fluorometry for quantitation, fluorescence microscopy for qualitative analysis, or flow cytometry for quantitation.
 

The DePsipher™ Kit uses a unique cationic dye (5,5’6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine iodide) to indicate the loss of the mitochondrial potential. 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™ remains in the cytoplasm as a green fluorescent monomeric form. Apoptotic cells, showing primarily green fluorescence, are 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.

There are several analysis compatible with etramethylrhodamine ethyl ester. Researchers use MitoShift (TMRE) to evaluate shifts in the delta-psi at the single mitochondrion level, by confocal microscopy. As the mitochondrial potential collapses, there is an outward flow of the dye along the altered pH gradient, leaving the mitochondria fluorescence free.

MitoShift can also be used in conventional fluorescence microscopy. The dye appears associated with mitochondria in healthy cells, generally in the perinuclear as a red-orange punctate fluorescence.

MitoShift can also be used in conventional fluorescence microscopy. The dye appears associated with mitochondria in healthy cells, generally in the perinuclear as a red-orange punctate fluorescence.

Finally, in late apoptotic cells or cells that have lost their cellular membrane integrity, the dye is released in the media and the fluorescence is lost. This feature allows the use of MitoShift with flow cytometry to discriminate necrotic or late apoptotic cells from healthy cells.