Caspase glo 8 and 9 kits

The activities of Caspase 8 and 9 were examined using bioluminescent Caspase-Glo® 8 and 9 kits (Promega, USA) following the manufacturer’s instructions. In brief, MCF-7 cells treated with AKBA (50, 100 and 200 µg mL⁻¹) for 24 h were subjected to 100 µL of Caspase-Glo® reagent for half an hour at room temperature. The luminescence of each sample was calculated using an ELISA reader (Bio-Rad, USA) at 405 nm. The values are represented mean ± SD and performed in triplicates, and treated samples were compared with positive and negative vehicles.

The apoptosis rate was assessed using a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA) employing Annexin V (Annexin V, Alexa Fluor 488 conjugate, Invitrogen, Carlsbad, CA, USA) and 7AAD (Bio Legend, San Diego, CA, USA) according to the manufacturer’s instructions [15 (link)]. A total of 10,000 events were collected. The data were analyzed using FlowJo software version 10.4 (TreeStar Inc., Ashland, OR, USA). Actinomycin D (Sigma-Aldrich, 80 μg/mL) was used as a positive control.
The activation of caspases 8 and 9 was evaluated with Caspase-Glo 8 and 9 kits (Promega, Madison, WI, USA) according to the manufacturer’s instructions [16 (link)]. K562 cells (6 × 10⁴/ well) were seeded in white 96-well plates and incubated with IC₅₀ or vehicle in the serum-free medium for 12 or 24 h. The luminescence was detected using a Varioskan spectrophotometer (Thermo Scientific). Actinomycin D (Sigma-Aldrich, 80 μg/mL) was used as a positive control.