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首页> 美国卫生研究院文献>other >Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle migration and lineage selection
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Live-cell time-lapse imaging and single-cell tracking of in vitro cultured neural stem cells – Tools for analyzing dynamics of cell cycle migration and lineage selection

机译：体外培养的神经干细胞的活细胞延时成像和单细胞跟踪–分析细胞周期迁移和谱系选择动力学的工具

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摘要

Neural stem cell (NSC) cultures have been considered technically challenging for time-lapse analysis due to high motility, photosensitivity, and growth at confluent densities. We have tested feasibility of long-term live-cell time-lapse analysis for NSC migration and differentiation studies. Here, we describe a method to study the dynamics of cell cycle, migration, and lineage selection in cultured multipotent mouse or human NSCs using single-cell tracking during a long-term, 7–14 day live-cell time-lapse analysis. We used in-house made PDMS inserts with five microwells on a glass coverslip petri-dish to constrain NSC into the area of acquisition during long-term live-cell imaging. In parallel, we have defined image acquisition settings for single-cell tracking of cell cycle dynamics using Fucci-reporter mouse NSC for 7 days as well as lineage selection and migration using human NSC for 14 days. Overall, we show that adjustments of live-cell analysis settings can extend the time period of single-cell tracking in mouse or human NSC from 24–72 h up to 7–14 days and potentially longer. However, we emphasize that experimental use of repeated fluorescence imaging will require careful consideration of controls during acquisition and analysis.
机译：由于具有高的运动性，光敏性以及在汇合密度下的生长，神经干细胞（NSC）培养对于延时分析在技术上具有挑战性。我们已经测试了用于NSC迁移和分化研究的长期活细胞时移分析的可行性。在这里，我们描述了一种方法，可在长期的7-14天活细胞时移分析中，使用单细胞跟踪研究培养的多能小鼠或人类NSC中细胞周期，迁移和谱系选择的动力学。我们在玻璃盖玻片培养皿上使用带有五个微孔的自制PDMS插入物，以在长期活细胞成像期间将NSC限制在采集区域。同时，我们已经定义了图像采集设置，用于使用Fucci-reporter小鼠NSC进行7天的单细胞跟踪细胞周期动力学，以及使用人NSC进行14天的谱系选择和迁移。总体而言，我们显示，活细胞分析设置的调整可以将小鼠或人类NSC中单细胞跟踪的时间从24-72小时延长至7-14天，甚至可能更长。但是，我们强调重复荧光成像的实验用途将需要在采集和分析过程中仔细考虑对照。

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