Epidermal growth factor (egf)

Colon cancer cell lines HCT116 (ATCC CCL-247), LoVo (ATCC CCL-229), RKO (CRL-2577), HCT15 (ATCC CCL-225), SW480 (ATCC CCL-228), SW620 (ATCC CCL-227), and T84 (ATCC CCL-248) were obtained from American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). All colon cancer cells were grown with ambient O₂ and 5% CO₂ at 37 °C. Immortalized, non-transformed human colonic epithelial cell lines (HCEC) were kindly provided by Jerry Shay (UT Southwestern) [18 (link)]. HCEC media consists of four parts DMEM to one-part media 199 (Sigma-Aldrich) supplemented with 1 μg/mL hydrocortisone, 25 ng/mL EGF, 10 μg/mL insulin, 5 nM sodium selenite, 2 μg/mL transferrin and 2% cosmic calf serum (GE Healthcare). HCECs were grown in 2% O₂ and 5% CO₂ at 37 °C within an enclosed hypoxia chamber. HCECs are grown on Corning™, Primaria™ plates.

Human EGF (Sigma Chemicals, St. Louis, MO, United States) was labeled with ¹²⁵I (Khlopin Radium Institute, Russia) using Iodogen (1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril, Sigma, United States). For labeling, 10 μg of the protein and 20–40 MBq of radioiodide in 0.05 M sodium borate buffer (pH 8.5) were incubated in glass vials coated with 10 μg of Iodogen for 15 min at room temperature. The reaction was terminated by addition of tyrosine to final concentration 5 mM. Radioiodinated EGF was purified by gel filtration through a PD-10 column (GE Healthcare Life Science, Great Britain) that was eluted with phosphate-buffered saline (pH 7.5). The yield for the radioconjugation reaction was 70–80% and the initial specific activity of ¹²⁵I-iodoEGF ranging from 2.2 to 3.1 GBq/mg of protein.