The tracing of neuronal cell lineages is critical to our understanding of cellular diversity in the CNS. This protocol describes a fluorescence birth-dating technique to label, track and isolate isochronic cohorts of newborn cells in the CNS in vivo in mouse embryos. Injection of carboxyfluorescein esters (CFSEs) into the cerebral ventricle allows pulse labeling of mitotic (M phase) ventricular zone (VZ) progenitors and their progeny across the CNS, a procedure we termed FlashTag. Specificity for M-phase apical progenitors is a result of the somata of these cells transiently contacting the ventricular wall during this cell-cycle phase, exposing them to CFSE injected into the cerebrospinal fluid. Using this approach, the developmental trajectory of progenitors and their daughter neurons can be tracked. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments or isolated using FACS for cell culture or (single-cell) RNA sequencing. Multiple embryos can be labeled within 30 min. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS and the processing of labeled tissue for immunohistochemistry.

中文翻译：

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使用FlashTag对中枢神经系统中等时性细胞群进行体内脉冲标记。

神经元细胞谱系的追踪对于我们对中枢神经系统中细胞多样性的理解至关重要。该协议描述了荧光出生日期技术，以标记，跟踪和分离小鼠胚胎体内CNS中新生细胞的等时队列。将羧基荧光素酯（CFSEs）注入脑室可对中枢神经系统的有丝分裂（M期）心室区（VZ）祖细胞及其后代进行脉冲标记，我们将这一过程称为FlashTag。M期根尖祖细胞的特异性是这些细胞在此细胞周期阶段短暂接触心室壁的结果，使它们暴露于注射到脑脊液中的CFSE中。使用这种方法，可以追踪祖细胞及其子神经元的发育轨迹。标记的细胞可以离体或在固定的组织中成像，靶向进行电生理实验，或使用FACS分离进行细胞培养或（单细胞）RNA测序。可以在30分钟内标记多个胚胎。该染料可以保留数周，从而可以在出生后识别出标记的细胞。该协议描述了在子宫内注射时的标记程序，使用FACS分离活细胞以及对标记的组织进行免疫组织化学处理。