Culturing Human Colorectal Cancer Cell Lines

The human colorectal cancer cell lines used in this study (HT29-D4, Caco-2, and RKO) were growth in DMEM medium (Dulbecco’s modified Eagle’s medium, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (fetal bovine serum) and L-glutamine (2 mM). Non-essential amino-acids were also added to the medium used to maintain Caco-2 cells. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. The HT29-D4 cells were originally selected from HT29 colon adenocarcinoma cells by Fantini et al. [15 (link)]. The oxaliplatin-resistant cells (Rox) were selected as described in our previous work [10 (link)]. These cells were cultured in the same medium as their parental cells, supplemented with 2 μM oxaliplatin (LOHP, Sigma-Aldrich, Merck, Darmstadt, Germany).