FLICA® is a powerful method to assess caspase activity. FLICA® probes are cell permeant, noncytotoxic Fluorescent Labeled Inhibitors of CAspases that covalently bind with active caspase enzymes. We have developed a far-red excitation and emission spectra FLICA® 660 probe for the detection of cells bearing active caspases.

Apoptosis is an evolutionarily conserved process of programmed cell suicide. It is centered on a cascade of proteolytic enzymes called caspases that are triggered in response to pro-apoptotic signals. Like the majority of other proteases, caspases are synthesized as pro-form precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity. Active caspase enzymes consist of two large (~20 kD) and two small (~10 kD) subunits that non-covalently associate to form a two heterodimer, tetrameric active caspase. Once activated, caspases cleave protein substrates leading to the eventual disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. Mammalian caspases play distinct roles in both apoptosis and inflammation.

Our FLICA® 660 poly caspase inhibitor probe contains the preferred binding sequence for all caspases, Val-Ala-Asp (VAD). This preferred poly caspase tripeptide binding sequence is labeled at the amino terminus end with a far-red fluorescent 660 dye and linked at the carboxyl end to a fluoromethyl ketone (FMK) reactive entity. The resulting cell permeant, fluorescent molecule, 660-VAD-FMK, optimally excites at 660 nm and emits between 685-690 nm. A conventional red HeNe laser with a 633 nm excitation provides excellent excitation efficiency, enabling cells labeled with FLICA 660 to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities.

To use FLICA® , add it directly to the cell media, incubate, and wash. FLICA is cell-permeant and will efficiently diffuse in and out of all cells. If there is an active caspase enzyme inside the cell, it will covalently bind with FLICA® 660-VAD-FMK and retain the far-red fluorescent signal within the cell.

Unbound FLICA® will diffuse out of the cell during the wash steps. Apoptotic cells will retain a higher concentration of FLICA® and fluoresce brighter than non-apoptotic cells. There is no interference from pro-caspases or inactive forms of the enzyme. If the treatment is causing cell death via apoptosis, apoptotic cells will have an elevated level of caspase activity relative to non-apoptotic or negative control cells and fluoresce with FLICA® . After labeling with FLICA® , cells can be counter-stained with other reagents and fixed or frozen.