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.
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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DOI：
10.1016/j.ymeth.2017.10.003
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发表时间：
2018-01-15
期刊：
影响因子：
--
通讯作者：
Anderson AJ
中科院分区：
文献类型：
--
作者：
Piltti KM;Cummings BJ;Carta K;Manughian-Peter A;Worne CL;Singh K;Ong D;Maksymyuk Y;Khine M;Anderson AJ
神经干细胞（NSC）培养物由于具有高运动性、光敏性和融合密度生长，一直被认为是延时分析的技术挑战。我们已经测试了长期活细胞延时分析用于NSC迁移和分化研究的可行性。在这里，我们描述了一种方法来研究细胞周期，迁移和谱系选择在培养的多能小鼠或人类神经干细胞的动态使用单细胞跟踪在一个长期的，7-14天的活细胞延时分析。我们使用内部制造的PDMS插入物，其在玻璃盖玻片培养皿上具有五个微孔，以在长期活细胞成像期间将NSC限制在采集区域中。同时，我们已经定义了使用Fucci报告小鼠NSC进行7天的细胞周期动态的单细胞跟踪以及使用人NSC进行14天的谱系选择和迁移的图像采集设置。总的来说，我们表明，活细胞分析设置的调整可以将小鼠或人NSC中单细胞跟踪的时间从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使用将需要在采集和分析过程中仔细考虑控制。
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.
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DOI：
10.1042/bj20112040
发表时间：
2012-06-15
期刊：
The Biochemical journal
影响因子：
--
作者：
Hindley C;Philpott A
通讯作者：
Philpott A
影响因子：
64.5
作者：
Sakaue-Sawano, Asako;Kurokawa, Hiroshi;Miyawaki, Atsushi
通讯作者：
Miyawaki, Atsushi
影响因子：
23.9
作者：
Ravin, Rea;Hoeppner, Daniel J.;Mckay, Ronald D. G.
通讯作者：
Mckay, Ronald D. G.
DOI：
10.3791/53247
发表时间：
2015-09-14
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Daynac M;Morizur L;Kortulewski T;Gauthier LR;Ruat M;Mouthon MA;Boussin FD
通讯作者：
Boussin FD
影响因子：
2.9
作者：
Yokose, Jun;Ishizuka, Toru;Yawo, Hiromu
通讯作者：
Yawo, Hiromu