ApoFluor® Red Apoptosis Detection Kits use a novel approach to detect active caspases. The methodology is based on a Fluorochrome Inhibitor of Caspases. Once inside the cell, the ApoFluor® Red inhibitor binds covalently to the active caspase8. These inhibitors are cell permeable and non-cytotoxic. For kits using red fluorescence, a sulforhodamine-labeled fluoromethyl ketone peptide inhibitor of caspases is used (ICN/ESP also offers a line of green apoptosis detection kits that use carboxyfluorescein-labeled inhibitors; please contact ICN/ESP for more details).
The ApoFluor® Red kit contains a fluorescent-labeled inhibitor from Enzyme Systems, either SR-VAD-FMK (sulforhodaminyl-L- valylalanylaspartyl fluoromethyl ketone) or SR-DEVD-FMK (sulforhodaminyl-L-valylalanylaspartyl fluoromethyl ketone). VAD is an amino acid sequence targeted by all caspases (VAD enters the cell and irreversibly binds to all activated caspases), while DEVD is targeted by caspase-3 and caspase-3 like caspases (DEVD enters the cell and irreversibly binds to activated caspase-3 > caspase-8 > caspase-7 > caspase-10 > caspase-6 in the order of decreasing binding affinity). When added to a population of cells, the ApoFluor® Red probe enters each cell and covalently binds to a reactive cysteine residue that resides on the large subunit of the caspase heterodimer, thereby inhibiting further enzymatic activity. Because the ApoFluor® Red reagent is covalently coupled to the enzyme, it is retained in the cell, while any unbound ApoFluor® Red reagent will diffuse out of the cell and is washed away. The remaining red fluorescent signal is a direct measure of the number of active caspase enzymes that were present in the cell when the reagent was added. Cells that contain the bound ApoFluor® Red can be analyzed by 96-well-plate based fluorometry, and fluorescence microscopy.