Mammalian Cells

Human SKOV-3 and AU565 (epithelial ovary and breast cancer cell lines, respectively) were from the American Type Culture Collection (ATCC, Manassas, VA). Human A431 (epithelial squamous carcinoma) cells were from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany). A431 cells were cultured in RPMI 1640 (BE12-115F; Lonza, Basel, Switzerland) containing 10% heat-inactivated calf serum. SKOV-3 and AU565 cells were cultured in RPMI 1640, containing 10% heat-inactivated calf serum, 2% sodium bicarbonate (BE17- 613E; Lonza), 1% sodium pyruvate (BE13-115E; Lonza), and 0.5% glucose (G8769; Sigma-Aldrich, St. Louis, MO). Cell lines were tested for mycoplasma contamination, and dedicated cell banks were prepared from early passage cultures.

For the transient expression of human EGFR in CHO-S cells (CHOs-EGFR) using the Freestyle MAX CHO Expression System (Life Technologies, Grand Island, NY), the codon-optimized (GeneArt; Life Technologies Europe BV, Bleiswijk, the Netherlands) coding sequence of human EGFR was cloned into pcDNA3.3 (Life Technologies).

EGFR, HER2, and TF cell surface expression was quantified using the QiFi flow cytometry bead-based indirect immunofluorescence kit (Dako, Glostrup, Denmark). Secondary conjugate F(ab′)₂ fragments of goat anti–mouse immunoglobulins conjugated with FITC (F0479; Dako) were used according to the supplier’s guidelines. As primary antibodies, mouse anti–human EGFR (555996; BD Biosciences, San Jose, CA), mouse anti–human HER2 (MAB1129; R&D Systems, Minneapolis, MN), and mouse anti–human TF (CLB, MW1838; Sanquin, Amsterdam, the Netherlands) were used for the quantification of human EGFR, HER2, and TF, respectively.