Ac-DEVD-AMC (AC-ASP-MET-GLN-ASP-7-AMINO-4-METHYLCOUMARIN) is the substrate of Caspase-3 .

The AMC moiety is highest at 2 h with an area?of 5.6±1.8, the AMC peak area for Ac-DEVD-AMC incubated without a lung specimen (substrate alone) for 6 h is relatively small [1]. Ac-DEVD-AMC can be used to monitor intracellular caspase-3 activity[2].

Lung fragments (about 20 mg each) are collected from rats and mice. The samples are incubated at 37°C in 50 mL KH buffer or MEM (continuously gass with 95% O2: 5% CO2) for up to 6 h. At specific time points, samples are incubated in oxygenated KH buffer or MEM with 32 μM zVAD-fmk or 15 μL DMSO for 20 min (f/v=1.0 mL). Ac-DEVD-AMC (37 μM) is then added and the incubation continues for an additional 20 min. At the end of incubation, the tissue is disrupted by vigorous homogenization for 2 min, sonication for 3 min, and 10 passages through a 27-G needle. This disruption procedure quenches the Ac-DEVD-AMC cleavage reaction due to dilution. The supernatants are collected by centrifugation (~6,300g for 90 min) through a microcentrifuge filter, separated on HPLC, and analyzed for the fluorogenic AMC moiety [2].