Cammeraat Maxime, Popovic Marko, Stam Wendy, Margadant Coert
Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam University Medical Center, Amsterdam, The Netherlands.
Department of Molecular Cell Biology and Immunology, Amsterdam University Medical Center, Amsterdam, The Netherlands.
Methods Mol Biol. 2023;2608:365-387. doi: 10.1007/978-1-0716-2887-4_21.
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.
集体细胞迁移对于多种病理生理过程至关重要，包括胚胎发育、伤口愈合、癌侵袭和芽生血管生成。迁移过程中领头细胞和跟随细胞的行为高度动态，涉及由肌动蛋白细胞骨架介导的广泛细胞形态变化。要随时间对这些快速动态变化进行成像，需要表达荧光蛋白和/或用荧光探针进行活细胞标记，然后在短时间间隔内采集一系列图像堆栈。这带来了与染料细胞毒性、信号损失相关的重大挑战，尤其是反复照射导致的光毒性，特别是在为多种染料使用单独通道以及在短时间间隔内对大z堆栈进行成像时。在本章中，我们介绍了对原代人内皮细胞群体进行多色活细胞标记的方法，随后在二维和三维蛋白质基质中进行多位延时成像。这些方法可以与RNA干扰结合使用以抑制特定蛋白质的表达，也可以在使用差异标记细胞群体混合物的镶嵌试验中进行。最后，我们提出了一种在低激光强度下进行长期成像以最小化激光诱导的细胞损伤的方案，随后使用人工智能进行成像后信号增强。
