2.7. Caspase-3 Activity Assay

Caspase-3 activity was measured using a fluorometric activity assay kit (Molecular probe, Eugene, OR, USA) following the manufacturer’s protocol. The substrate, rhodamine 110 bis-(N-CBZ-l-aspartyl-l-glutamyl-l-valyl-l-aspartic acid amide) (Z-DEVD-Rho 110), was cleaved proteolytically by caspase-3. After 24 h treatment with ACA (0.88, 1.17, and 1.75 µM corresponding to IC₂₀, IC₃₀ and IC₄₀, respectively), the cells were harvested and lysed in 1 mL lysis buffer. The sample was incubated with the substrate Z-DEVD-Rho 110. Fluorescence intensity was determined using a LightCycler 480^® (Roche Diagnostic, Inc., Mannheim, Germany) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. Values were expressed as arbitrary units of Rhodamine-110 (Rho 110) fluorescence.