Caspase-3 Activation Assay by Flow Cytometry

The activation of caspase-3 was evaluated via flow cytometry according to the method described by Moraes et al. (2013 (link)), with minor modifications. Cells were seeded in 24-well plates (10⁵ cells/mL), where they were cultured in medium containing 10% FBS and treated with IC₅₀ of the AECL or AECR, for 24 h. After treatment, the cells were centrifuged, washed, and fixed in 2% paraformaldehyde in PBS for 30 min. The cells were then washed with glicin (0.1 M) in PBS, permeabilized with 0.01% saponin for 30 min, and blocked in PBS containing 1% BSA for 30 min at room temperature. Subsequently, the cells were incubated with an anti-active-caspase-3 monoclonal antibody conjugated with FITC (BD-Pharmingen, San Diego, CA, USA) in the dark at room temperature for 40 min. After incubation, fluorescence was analyzed in a FACScalibur Flow Cytometer (Becton Dickinson, USA) using CellQuest software (10,000 events were collected per sample).