Caco2 human colorectal adenocarcinoma cells

DiFi human colorectal adenocarcinoma cells and a cetuximab-resistant DiFi subline, DiFi5, were described previously [24 (link)–27 (link)]. DiFi-AG, a DiFi subline with acquired resistance to AG1478, an EGFR TKI, was generated by chronic exposure to serially increased doses of AG1478 (from 0.1 μM to 5 μM) for over 1 year. Caco2 human colorectal adenocarcinoma cells were purchased from American Type Culture Collection. All cell lines were maintained in a mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1, v/v) supplemented with 10% fetal bovine serum in a 37°C humidified atmosphere containing 95% air and 5% CO₂ and were split twice a week.
To measure cell proliferation following treatments of cells in 24-well plates, 10 mg/mL methylthiazolyldiphenyl-tetrazolium bromide (MTT) was added (50 μL in 0.5 mL medium/well), and the cells were incubated for an additional 2 h. The cells were then lysed with a lysis buffer (500 μL/well) containing 20% sodium dodecyl sulfate in dimethyl formamide/H₂O (1:1, v/v; pH 4.7) at 37°C for at least 6 h. The relative number of surviving cells in each group was determined by measuring the optical density (OD) of the cell lysates at an absorbance wavelength of 570 nm. The OD value of each treatment group was expressed as a percentage of the OD value of the untreated control cells [36 (link)].