Abstract
Collective cell migration is crucial for a variety of pathophysiological processes including embryonic development, wound healing, carcinoma invasion, and sprouting angiogenesis. The behavior of leading and following cells during migration is highly dynamic and involves extensive cellular morphological changes mediated by the actin cytoskeleton. Imaging these rapid and dynamic changes over time requires expression of fluorescent proteins and/or live labeling with fluorescent probes, followed by acquiring series of image stacks at short intervals. This presents significant challenges related to dye cytotoxicity, signal loss, and in particular phototoxicity resulting from repeated irradiation, especially when using separate channels for multiple dyes and when imaging large z-stacks at short time intervals. In this chapter, we present methods for multicolor live-cell labeling of primary human endothelial cell populations, followed by multi-position time-lapse imaging in 2D and in 3D protein matrices. These approaches can be performed in combination with RNA interference to suppress the expression of specific proteins, as well as in mosaic assays using mixtures of differentially labeled cell populations. Finally, we present a protocol for long-term imaging at low laser intensity to minimize laser-induced cell damage, followed by post-imaging signal enhancement using artificial intelligence.
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Acknowledgments
We are grateful to Luc Reymond (Spirochrome, Switzerland) for his kind gifts of probes and advice. We further acknowledge Joost Boex (Tebu-bio, the Netherlands) for organizing probe delivery, Jeroen Kole (Confocal.nl, Amsterdam, the Netherlands) for advice, and Nanne Paauw (Microscopy and Cytometry core facility Amsterdam UMC, location VUmc) for technical support with imaging.
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1 Electronic Supplementary Movie Legends
Live imaging of nuclear and cytoskeletal dynamics during endothelial cell migration in a scratch assay (AVI 14820 kb)
Live imaging of nuclear and cytoskeletal dynamics in a mosaic endothelial cell population during cell migration (AVI 14363 kb)
Live imaging of nuclear and cytoskeletal dynamics in a sprouting endothelial tip cell. (AVI 14820 kb)
Phototoxicity upon live-cell imaging of sprouting tip cells (AVI 14820 kb)
Signal enhancement in live-cell movies using AI-based image restoration (AVI 14820 kb)
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Cammeraat, M., Popovic, M., Stam, W., Margadant, C. (2023). Live-Cell Labeling and Artificial Intelligence Approaches for High-Resolution XYZT Imaging of Cytoskeletal Dynamics During Collective Cell Migration. In: Margadant, C. (eds) Cell Migration in Three Dimensions. Methods in Molecular Biology, vol 2608. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2887-4_21
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DOI: https://doi.org/10.1007/978-1-0716-2887-4_21
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