Chapter 2 │ Page 40 culture. In summary, this study provides clear insights into the pathophysiological consequences of accumulating photodamage after viral transduction of fluorophores into cancer cells and suggests a direct link with increased sensitivity towards NTP in vitro. Therefore, this e ect must be carefully considered and addressed when evaluating NTP therapy response and other redox-based therapies, in order to delineate confounding interactions and accurately draw conclusions. 2 EXPERIMENTAL METHODS In this work, we aimed to elucidate the e ect of fluorescently-labelled viral transduction on phototoxicity and cellular response to NTP therapy. The SC263 squamous cell carcinoma cell line was used and treated with a microsecondpulsed dielectric barrier discharge (DBD) plasma system. Experiments were performed at low and high passages to further determine the e ect of prolonged cell culturing. For all experiments, commercially available human cell lines were used and no ethical approvals were necessary. The SC263 cell line was kindly provided by Prof. Dr. Sandra Nuyts (University Hospital Leuven, Leuven, Belgium). 2.1 Cell Culture and Transduction The human head and neck squamous cell carcinoma cell line, SC263, was cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with fetal bovine serum (FBS) (10%), L-glutamine (1%), and penicillin/ streptomycin (1%). Cells were incubated in a humidified atmosphere at 5% CO2 and 37°C. Following our strict culturing method, cells were sub-cultured at 80% confluence and passaged at a 1:5 ratio every third day of culturing. Cellular autofluorescence was not observed throughout the experiments. IncuCyte® NucLight Red Lentivirus Reagent (promoter: EF1a; fluorophore: mKate2; antibiotic selection: Puro) (4627, Essen Bioscience, Newark, United Kingdom) was
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