Hanne Verswyvel

Chapter 2 │ Page 45 exposure time (Blue: 500ms; Green: 400ms), and led intensity (Blue: 60%, Green: 50%) were automatically determined with the live viewer function. Analysis masking parameters for the 2 fluorescence signals encompassed a sensitivity of 90%, with object width and length minima and maxima at 5 and 30 µm, respectively. Cell survival rates were calculated for each well (Equation 2.1). Live cell counts were determined by subtracting the measured dead cell counts (Green counts) from the total cell counts (Blue count). The live cell counts were then normalized to the mean of the total cell counts of the untreated condition to give the percentage of cell survival. (%) = ( − ) ∗ 1 0 0 (2.1) 2.4 Proliferation Analysis Untreated SC263 and SC263T cells were seeded into 96-well plates at 5 × 104 cells/mL (200 µL/well) to determine their baseline growth rate. Plates were monitored for 72 hours in the temperature- and CO2- controlled Incucyte ZOOM® live-cell imaging system (37oC and 5% CO2). Phase contrast images (10x) were captured every 2 hours and analyzed using the Incucyte ZOOM® software to determine confluence as an indicator of cell proliferation. All images per experiment were analyzed together using the same image analysis parameters to reduce variability. 2.5 Intracellular ROS Analysis In order to determine the baseline redox status of untreated SC263 and SC263T cells, the intracellular ROS levels were measured. Cells were seeded into 96-well plates at 5 × 104 cells/mL (200 µL/well) and incubated at 37oC and 5% CO2 overnight to allow for cell attachment. Following incubation, the medium was removed and replaced with fresh medium containing 2.5 µM CellROXTM Green Reagent (C10444,

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