Chapter 2 │ Page 56 fluorochrome selection, reduction of scanning intensity, and limiting the number of image acquisition events would limit laser-induced phototoxicity during timelapse experiments. Another option is to opt for the use of transient fluorescence dyes, where cells are only tagged before an experiment and not during maintenance of the cell culture. Although the fluorescence expression from this incorporation method is typically less stable compared to more integrated techniques, such as viral transduction, it helps reduce the acquired biological variance from potential prolonged phototoxicity. These considerations should not only be taken into account for experiments utilizing 2D monolayer cultures demonstrated here, but also for those using 3D in vitro models. Moreover, immunofluorescence is also used in in vivo research for tracking tumor growth in orthotopic models and follow-up of metastasis[46, 47]. Although ambient light is not a confounding factor in situ in the in vivo environment, our findings could have important implications to take into account during preparatory cell culturing and experimental handling prior to tumor cell inoculation.
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