Caspase glo 8 and 9 assays

Cells were seeded into white 96-well plates (Fisher Scientific, Loughborough, UK) at 2.5 × 10³ cells per well and treated with two alkylating agents: cisplatin and cyclophosphamide. These alkylating agents were selected as they showed a greater synergistic effect when used in combination with polyphenols; across both lymphoid and, myeloid leukaemia cell lines. These alkylating agents were used alone and in combination with those polyphenols that previously showed a synergistic effect and were used at their LSDs for 24 h, together with a 0.1% (v/v) ethanol vehicle control. Following treatment Caspase-Glo^® 8 and 9 assays were used as per manufacturerʼs instructions (Promega, Southampton, UK) to determine caspase 8 and 9 activity. Luminescence was measured using a Wallac Victor 2 1420 and were normalised to vehicle controls.

Cells were seeded in cell culture medium in 96-well plates to adhere overnight. Cells were treated with the indicated reagents for 24 h and active caspases-8 or -9 were detected using Caspase-Glo 8 and 9 assays (Promega) according to the manufacturer's protocol.

Cells were seeded into white 96-well plates at 2.5×10³ cells per well and treated with each topoisomerase inhibitor and polyphenols alone and in combination at their LSD for 24 h, together with a 0.1% (v/v) vehicle control. Following treatment Caspase-Glo 8 and 9 assays were used as per manufacturer's instructions (Promega) to determine caspase 8 and 9 activity. Luminescence was measured using a Wallac Victor 2 1420 (Perkin Elmer Coventry, UK) and was normalised to vehicle controls.