Phototoxicity and cell passage affect intracellular reactive oxygen species levels and sensitivity towards non-thermal plasma treatment in fluorescently-labeled cancer cells

Live-cell imaging with fluorescence microscopy is a powerful tool, especially in cancer research, widely-used for capturing dynamic cellular processes over time. However, light-induced toxicity (phototoxicity) can be incurred from this method, via disruption of intracellular redox balance and an overload of reactive oxygen species (ROS). This can introduce confounding effects in an experiment, especially in the context of evaluating and screening novel therapies. Here, we aimed to unravel whether phototoxicity can impact cellular homeostasis and response to non-thermal plasma (NTP), a therapeutic strategy which specifically targets the intracellular redox balance. We demonstrate that cells incorporated with a fluorescent reporter for live-cell imaging have increased sensitivity to NTP, when exposed to ambient light or fluorescence excitation, likely through altered proliferation rates and baseline intracellular ROS levels. These changes became even more pronounced the longer the cells stayed in culture. Therefore, our results have important implications for research implementing this analysis technique and are particularly important for designing experiments and evaluating redox-based therapies like NTP.

Automated summary

Live-cell imaging with fluorescence microscopy is a powerful tool in cancer research but can result in phototoxicity, causing disruption of redox balance and an overload of reactive oxygen species. We investigated the impact of phototoxicity on cellular homeostasis and response to non-thermal plasma (NTP), revealing increased sensitivity to NTP in cells used for live-cell imaging, likely due to altered proliferation rates and ROS levels. These findings have important implications for research using this technique and evaluating redox-based therapies.