4.5. Apoptosis Assay

Apoptosis was assessed by measuring the caspase 3 activity using a colorimetric caspase-3 activity assay (Sigma-Aldrich). Briefly, DLD-1 2N and 4N cells were treated with 20 µM of oxaliplatin, 25 µM of 5-fluorouracil or FOLFOX. RKO, SW837 and RPE1 cell lines were treated with FOLFOX. After 24 h, cells were lysed in lysis buffer and protein concentrations were determined by using the Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Same amounts of each protein extract (100 µg) were assayed with the colorimetric caspase-3 substrate Ac-DEVD-pNA. The release of the yellow chromophore p-nitroanilide (pNA) was measured in an Epoch Microplate Spectrophotometer (BioTek) at 405 nM. Caspase-3 activity was calculated in comparison to a pNA standard curve.