Ac-DEVD-AMC (Synonyms: NAcetylAspGluValAsp7amido4Methylcoumarin, Caspase3 Substrate (Fluorogenic))

Cell experiment:

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 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 microcentrifuge filter, separateed on HPLC, and analyzed for the fluorogenic AMC moiety[2].

References:

[1]. Ahmed R Alsuwaidi, et al. Bioenergetics of murine lungs infected with respiratory syncytial virus. Virol J. 2013; 10: 22.
[2]. Ahmed R Alsuwaidi, et al. Lung tissue bioenergetics and caspase activity in rodents. BMC Res Notes. 2013; 6: 12.