Caspase-3 Activity Assay in U937 Cells

Caspase-3 activity was analyzed using a colorimetric caspase-3 detection kit (Sigma) and a published protocol (Winstel et al., 2018 (link); Tantawy et al., 2022 (link)). Briefly, U937 cells or U937 MФ along with appropriate controls were pre-incubated for 2 h at 37°C under 5% CO₂ in RPMI 1640 medium containing 10% hi-FBS and 1 μM of (R)-DI-87. Controls received vehicle only. Subsequently, cells were exposed to various concentrations of either dAdo or dGuo (100 μM for U937; 200 μM for U937 MФ) and incubated for 24 h at 37°C under 5% CO₂ in RPMI 1640 growth medium. Cells were collected via centrifugation (U937) or a detachment-centrifugation step (U937 MФ) using trypsin-EDTA solution and washed once in PBS. Next, 1.0 × 10⁷ cells were lysed in pre-chilled lysis buffer (Sigma kit) for 20 min. This step was performed on ice. Resulting lysates were centrifuged at 4°C (18,000 × g for 10 min) to obtain cell- and debris-free supernatants which were incubated with the caspase-3 substrate Ac-DEVD-pNA according to the manufacturer’s instructions. Caspase3 activity was determined based on the amount of released pNA that can be detected at 405 nm.