Ctb assay

The sterilized and washed constructs were placed in the middle of a 24-well plate and 8000 cells per well were seeded. As control the same cell number per square centimeter was seeded in wells without constructs. After one day, the constructs were moved in a new well plate, where the cells grew only on the surface of the construct. After one, three, and seven days of cultivation the viability of the cells was evaluated by CTB assay (Promega, Mannheim, Germany) and calcein-acetoxymethyl (AM, Merck, Darmstadt, Germany) and ethidium-homodimer (Sigma Aldrich, München, Germany) staining. CTB assay was performed according to the information provided by the manufacturer. Briefly, a 10% CTB solution was prepared with α-MEM (1:10 v/v) and 500 µL were added to each well after carefully aspirating the old medium. Additionally, wells without cells were filled with the 10% CTB solution and used as a blank. The fluorescence was measured with a microplate reader (Fluoroskan Ascent, Thermo Fisher Scientific Inc., Waltham, MA, USA) at an extinction wavelength of 544 nm and an emission wavelength of 590 nm after 4 h of incubation at 37 °C. To each well 500 µL of a staining solution with 4 µM calcein-AM, 4 µM ethidium-homodimer in α-MEM were added and incubated for 45 min at 37 °C and evaluated with a Cytation 5-Cell Imaging Multi-Mode Reader (Biotek Instruments, Winooski, VT, USA).

ADCC was performed by incubation of NK cells with tumor cells in an E : T ratio of 10 : 1 in the presence of anti-EGFR mAbs. After 24 h of coculture, plates were carefully washed, and a three-hour cell titer blue (CTB) assay (Promega, Leiden, the Netherlands) was performed according to the manufacturer's protocol. Readout was performed on a Bio-Rad model 680 microplate reader (Bio-Rad, Hercules, CA). Percentage killing was calculated by 100—percentage of remaining tumor cells.