Caspase glotm 3 7

Caspase-3 and -7 activities were assessed using a commercial kit (Caspase-Glo^TM 3/7, Promega, Fitchburg, USA) as per the manufacturer’s instructions. Briefly clarified supernatants (50 μL) were diluted according to individual protein concentrations to yield final protein concentrations of 0.5 mg protein/mL. These protein-adjusted samples were then added to Caspase-Glo^TM 3/7 reagent (1:1 v/v, 20 °C for 60 min) and luminescence measured using a FLUOstar Omega reader (BMG LABTECH, Offenburg, Germany). Total caspase 3/7 activity was represented as mean luminescence after 60 min of monitoring.

The activities of caspase-3/7, -8 and -9 were measured using a luminescence assay, with Caspase-Glo^TM 3/7, Caspase-Glo^TM 8 and Caspase-Glo^TM 9 assay kits (Promega, USA). The assay was conducted following the manufacturer’s protocols. Briefly, HepG2 cells were cultured in a 96-well plate and incubated for 24 h in a humidified atmosphere of 5% CO₂ at 37 °C. The cells were then treated with sulforaphene (in serum free media) at an IC₅₀ concentration of 72 h (33.8 μM). Cells were then incubated for 24, 48 and 72 h. After treatment, 100 μL of assay reagent was added to each well and incubated for 1 h at room temperature. Finally, the luminescence was measured using a microplate reader (Tecan Infinite M 200 PRO, Männedorf, Switzerland).