Caspase-3/-7 detection assay

Caspase-3/-7 activity was quantitated using the Caspase-Glo 3/7 Assay (Promega G8090) according to the manufacturer’s instructions. The Beckman Coulter DTX 880 multimode plate reader was used to query the processed samples.

Cellular reactive oxygen species detection. This assay utilized the cell permeant, fluorogenic dye 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFDA) to measure cellular production of reactive oxygen species (ROS). In the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent form, which is later oxidized by ROS into the highly fluorescent 2ʹ,7ʹ-dichlorofluorescin (DCF). The excitation/emission spectrum of DCF is 495/529nm, respectively, which was measured using at Ex/Em 485/535 on a Beckman Coulter DTX 880 multimode reader. This assay was obtained from Abcam (ab113851) and carried out according to the manufacturer’s instructions. As a positive control, cells were treated with 100 µM tert-butyl hydrogen peroxide (TBHP) to induce DCF fluorescence.