Ac-DEVD-AFC, a fluorogenic substrate, undergoes cleavage by caspase-3, producing a blue fluorescent compound upon excitation at 400 nm, which can be quantified at an emission maximum of 505 nm.

Caspase activation was measured using the fluorogenic compound N-acetyl-asp-glu-val-asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC). We show that this substrate rapidly enters cells where it is efficiently cleaved at the aspartate residue by specific caspases, yielding the fluorescent compound 7-amino-4-trifluoromethyl coumarin (AFC). Following cell disruption, released AFC was separated on HPLC and detected by fluorescence. The appearance of AFC in cells was blocked by the pancaspase inhibitor benzyloxycarbonyl-val-ala-asp-fluoromethylketone, thus establishing that intracellular caspases were responsible for the cleavage. Caspase activity was first noted after about 2 h of incubation with doxorubicin or dactinomycin, the production of AFC being linear with time afterward[1].