The Image-iT LIVE red and green caspase detection kits for microscopy employ a novel approach to detecting active caspases: both assay kits take advantage of a fluorescent inhibitor of caspases (FLICA) methodology, which is an affinity label. This reagent 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), and aspartic acid-glutamic acid-valine-aspartic acid (DEVD) for caspase 3/7. A sulforhodamine (SR) group or 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 red or green 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 Image-iT LIVE Red Poly Caspases, Red Caspase-3/7, Green Poly Caspases, and Green Caspase-3/7 Detection kits are SR-VAD-FMK, SR-DEVD-FMK, FAM-VAD-FMK, and FAM-DEVD-FMK, respectively.