Chapter 2 │ Page 42 The optimal confluence on the day of NTP treatment is between 70-75%, and the best seeding density was determined for all groups based on their growth rates (Figure 1b-e). Here, cells were seeded at di erent densities and phase-contrast imaging was performed over 48 hours using the Incucyte ZOOM imaging system (Sartorius, Germany). Based on these measurements, 2.5 × 104 SC263 cells and 3.0 × 104 SC263T cells were seeded into each well, 1 day prior to NTP treatment. Figure 1: Cellular growth curves. Fluorescently-transduced cells were seeded into 96well plates at di erent densities and monitored over 48 hours to determine growth rates and optimal seeding density before NTP treatment. (a) IncuCyte® NucLight Red Lentiviral transduction and puromycin selection provided homogeneous and stable red fluorescence nuclear expression as seen in the phase contrast (left), fluorescence (middle), and merged (right) images. Images were taken by the Incucyte ZOOM® at 10x and the scale bars represent 100 µm. Confluence of non-transduced SC263 cells at (b) low passages (PL) and (c) high passages (PH) as well as transduced cells (SC263T) at (d) PL and (e) PH were also determined via live-cell image analysis using the Incucyte ZOOM® imaging system. 2.2 NTP Treatment A microsecond-pulsed DBD system, described in our previous studies [27-30], was used for all treatments. This system consists of a microsecond-pulsed power supply (Megaimpulse Ltd, Russia) and a round-bottom dielectrically covered electrode, providing an electrically and thermally stable plasma [31]. All electrical and operation parameters are described in Table 1. The energy per pulse of NTP,
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