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 对中枢神经系统中的等时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了解中枢神经系统细胞多样性至关重要。该协议描述了一种荧光出生约会技术，用于标记、跟踪和分离小鼠胚胎体内中枢神经系统中新生细胞的等时队列。将羧基荧光素酯（CFSE）注射到脑室中可以对有丝分裂（M期）心室区（VZ）祖细胞及其整个中枢神经系统的后代进行脉冲标记，我们将这一过程称为FlashTag。 M期心尖祖细胞的特异性是由于这些细胞的体体在该细胞周期阶段短暂接触心室壁，使它们暴露于注射到脑脊液中的CFSE的结果。使用这种方法，可以追踪祖细胞及其子代神经元的发育轨迹。标记的细胞可以离体或在固定组织中成像，针对电生理学实验或使用 FACS 进行分离以进行细胞培养或（单细胞）RNA 测序。 30 分钟内即可对多个胚胎进行标记。染料可保留数周，以便在出生后识别标记的细胞。该协议描述了使用子宫内注射的标记程序、使用 FACS 分离活细胞以及用于免疫组织化学的标记组织的处理。