FLICA Apoptosis Detection Kits use a novel approach to detect active caspases. The methodology is based on a Fluorochrome Inhibitor of Caspases (FLICA). Once inside the cell, the FLICA inhibitor binds covalently to the active caspase⁵. These inhibitors are cell permeable and non-cytotoxic. For detection using red fluorescence, a sulforhodamine-labeled fluoromethyl ketone peptide inhibitor of caspases is used. ICT also offers Carboxyfluorescein FLICA assays that use green fluorescence.

The red FLICA kits contain a fluorescent-labeled inhibitor, either SR-VAD-FMK (sulforhodaminyl-L-valylalanylaspartyl fluoromethyl ketone), or SR-DEVD-FMK (sulforhodaminyl-L-aspartylglutamylvalylaspartylfluoro-methyl ketone). VAD is an amino acid sequence targeted by all caspases, while DEVD is targeted by caspase-3. When added to a population of cells, the FLICA 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 FLICA reagent is covalently coupled to the enzyme, it is retained in the cell, while any unbound FLICA 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 FLICA can be analyzed by 96-well-plate based fluorometry and fluorescence microscopy.

Because the FLICA reagent SR-VAD-FMK irreversibly binds to many activated caspases (caspase-1, -3, -4, -5, -6, -7, -8 and -9), it can be used as a generic probe for the detection of most caspases. In comparison, when the FLICA reagent SR-DEVD-FMK enters the cell, it primarily binds to caspase-3, therefore it can be used to measure the amount of active caspase-3 that was present in the cell at the time when the SR-DEVD-FMK FLICA reagent was added.

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References:

Ekert, P.G., J. Silke, and D.L.Vaux. 1999. Caspase inhibitors. Cell Death and Differ. 6:1081-1086.