Sue & Bill Gross Stem Cell Center, University of California-Irvine, Irvine, CA 92697, USA; Physical & Medical Rehabilitation, University of California-Irvine, Irvine, CA 92697, USA; Institute for Memory Impairments & Neurological Disorders, University of California-Irvine, Irvine, CA 92697, USA.
Sue & Bill Gross Stem Cell Center, University of California-Irvine, Irvine, CA 92697, USA; Physical & Medical Rehabilitation, University of California-Irvine, Irvine, CA 92697, USA; Institute for Memory Impairments & Neurological Disorders, University of California-Irvine, Irvine, CA 92697, USA; Anatomy & Neurobiology, University of California-Irvine, Irvine, CA 92697, USA.
Methods. 2018 Jan 15;133:81-90. doi: 10.1016/j.ymeth.2017.10.003. Epub 2017 Oct 16.
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.
神经干细胞 (NSC) 培养物由于其高迁移率、光敏感性以及在汇合密度下的生长特性，在进行延时分析时技术上具有挑战性。我们已经测试了对 NSC 迁移和分化研究进行长期活细胞延时分析的可行性。在这里，我们描述了一种方法，用于使用单细胞跟踪在长期、7-14 天的活细胞延时分析中研究培养的多能性小鼠或人类 NSC 中的细胞周期、迁移和谱系选择的动力学。我们使用内部制造的 PDMS 插入物，在玻璃盖玻片培养皿上有五个微孔，将 NSC 限制在长期活细胞成像期间获取的区域内。同时，我们已经定义了用于使用 Fucci 报告小鼠 NSC 进行 7 天的细胞周期动力学单细胞跟踪以及使用人类 NSC 进行 14 天的谱系选择和迁移的图像采集设置。总体而言，我们表明，活细胞分析设置的调整可以将小鼠或人类 NSC 的单细胞跟踪时间从 24-72 小时延长到 7-14 天，甚至更长。然而，我们强调，重复荧光成像的实验应用将需要在采集和分析过程中仔细考虑对照。
