Live cell imaging is used to track the dynamic adaptation of cell size and motility to various external factors. Bright-field configuration can be used for these experiments; however, the analysis can be challenging and difficult to automate. In this direction, a superior alternative is represented by the use of live cell dyes, which provide intense fluorescence from subcellular structures of living cells. Yet, the potential chemo- and photo-toxicity of the fluorophores poses the necessity of an accurate protocol optimization to avoid artefacts. Toxicity studies generally focus on cell proliferation and apoptosis, neglecting the cellular activities under investigation. Here, we present the case of SYTO 13 in combination with primary endothelial cells. The optimization of the staining procedure is tested comparing cell proliferation and motility rate. In addition, the combined effect of staining and fluorescent illumination, reporting for photochemical toxicity, is evaluated. We demonstrate that while cell viability and proliferation are mainly unaffected by the staining and imagining protocols, a significant reduction of the motility rate is induced both by the chemical dye alone and in combination with fluorescent illumination. The general implications for this procedure are discussed.
© 2020 American Society for Photobiology.