Department of Neuroscience, University of Wisconsin-Madison.
Morgridge Institute for Research; Department of Biomedical Engineering, University of Wisconsin-Madison.
J Vis Exp. 2024 Apr 12(206). doi: 10.3791/63110.
Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensities and fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.
神经干细胞（NSC）通过一个称为成人神经发生的过程在成人大脑中分裂并产生新的神经元。成人 NSC 主要处于静止状态，这是一种可逆的细胞状态，它们已经退出细胞周期（G0），但仍然对环境有反应。在成人神经发生的第一步中，静止的 NSC（qNSC）接收到信号并激活，退出静止状态并重新进入细胞周期。因此，了解 NSC 静止和静止退出的调节剂对于未来针对成人神经发生的策略至关重要。然而，我们对 NSC 静止的理解受到识别静止的 NSC（qNSC）和激活的 NSC（aNSC）的技术限制的限制。本协议描述了一种通过成像 NSC 自发荧光来识别和富集体外培养中产生的 qNSC 和 aNSC 的新方法。首先，本协议描述了如何使用共聚焦显微镜识别 qNSC 和 aNSC 的自发荧光标记物，使用自发荧光强度来分类 NSC 的激活状态。其次，本协议描述了如何使用荧光激活细胞分选器（FACS）根据自发荧光强度来分类 NSC 的激活状态并富集 qNSC 或 aNSC 样本。第三，本协议描述了如何使用多光子显微镜以单细胞分辨率进行荧光寿命成像（FLIM），分类 NSC 的激活状态，并使用自发荧光强度和荧光寿命跟踪静止退出的动力学。因此，该协议为研究 NSC 静止和静止退出提供了一种活细胞、无标记、单细胞分辨率的工具包。
