Caspase 9 glo reagent

SGC-7901/DDP cells were seeded in a 96-well plate and starved overnight in RPMI 1640 medium with 0.5% serum. Being treated with ZJW of different concentration for 48 hours, 100 μL of Caspase-Glo 3 or Caspase-Glo 9 reagent (Promega, USA) was added to each sample and incubated at room temperature for another 2 hours. The luciferase activity was measured using a TD 20/20 luminometer (Promega, USA). Each sample was measured in triplicate.

Briefly, WM266–4 and M14 cells were plated in 384-well plates in 5 μL of complete media (EMEM and RPMI-1640, respectively). 5 μL of 200 μM 2155–14 and 2155–18 were added to the cells. Plates were incubated at 37 °C, 5% CO₂ and 95% relative humidity for various lengths of time. After incubation, 5 μL of caspase 3/7, caspase 8, and caspase 9 Glo® reagent (Promega cat#: G7570) were added to each well, and incubated for 15 min at room temperature. Luminescence was recorded using a Biotek Synergy H4 multimode microplate reader. In the case of caspase 6, 5 μL of 100 μM caspase 6 substrate AFC-VEID in lysis buffer was added and incubated for 1 h at 37 °C, 5% CO₂ and 95% relative humidity and fluorescence intensity was measured at λ_excitation = 400 nm and λ_emission = 505 nm using a Biotek Synergy H4 multimode microplate reader.