The Vybrant FAM caspase assay kits for flow cytometry employ a novel approach to detecting active caspases: the kits take advantage of FLICA methodology, which is an affinity label. This affinity label associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amino acid sequence. Different amino acid recognition sequences are used to detect different caspases: valine-alanine-aspartic acid (VAD) for poly caspases (including caspase-1, -3, -4, -5, -6, -7, -8, and -9), aspartic acid-glutamic acid-valine-aspartic acid (DEVD) for caspase-3/7, and leucine-glutamic acid-threonine-aspartic acid (LETD) for caspase-8. A carboxyfluorescein (FAM) group is attached as a reporter.

The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining fluorescent signal is a direct measure of the amount of active caspase present at the time the inhibitor was added. The approximate excitation and emission peaks of the FLICA, propidium iodide, and Hoechst 33342 reagents are 488 nm/⁄530 nm, 535⁄ nm/6617 nm, and 350 nm/⁄461 nm, respectively. The detection reagents included within the individual Vybrant FAM Poly Caspases Assay, Caspase-3 and -7 Assay, and Caspase-8 Assay apoptosis kits are 488⁄⁄530 FAM-VAD-FMK reagent, 488/⁄530 FAM-DEVD-FMK, and 488/⁄530 FAM-LETD-FMK, respectively.