Chapter 2 │ Page 51 transduction, cell proliferation was significantly reduced (Figure 5b). Indeed, even for low passages, fluorescence transduction a ected cell growth compared to that of the parental cells (SC263T-PL vs SC263-PL; p = 0.003). This e ect was even more pronounced the longer the cells stayed in culture (SC263T-PH vs SC263-PH; p < 0.0001). This was even more clearly evidenced as cell passage did not significantly a ect growth of non-transduced cell lines, while significant di erences were measured between low and high passaged transduced cell lines (SC263T-PL vs SC263T-PH; p < 0.0001). Taken together, it is clear that following fluorescent transduction, the proliferation rate was significantly a ected early after transduction and further reduced after continuous culturing. Here, it does not appear that the higher sensitivity to NTP treatment is due to a faster proliferation rate, as previously hypothesized in literature. Another prevalent hypothesis for why certain cells are more sensitive to NTP therapy is based on their ability to balance their cellular redox status [28, 29]. It has been hypothesized that certain cells have higher baseline intracellular ROS levels, and therefore delivery of additional ROS from NTP treatment leads to irreversible oxidative distress and damage. Therefore, we aimed to investigate whether the more sensitive SC263T-PH cells had elevated baseline intracellular ROS levels compared to their parental counterparts. Baseline ROS levels of the cell lines were evaluated with CellROXTM Green Reagent (2.5 µM) and a single-timepoint analysis. It is clear that the SC263T-PH cells exhibited higher fluorescence intensity compared to all other cell counterparts, thus indicating higher levels of intracellular ROS (Figure 5c). The mean fluorescence intensity was quantified using the Incucyte ZOOM® live-cell analysis software and fold changes were calculated between parental and transduced counterparts (Figure 5d), and revealed that baseline intracellular ROS levels in
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