Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland.
Interfaculty Institute of Bioengineering, EPFL, Lausanne, Switzerland.
Nat Protoc. 2018 Oct;13(10):2297-2311. doi: 10.1038/s41596-018-0038-1.
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.
追踪神经元细胞谱系对于理解中枢神经系统中的细胞多样性至关重要。本方案描述了一种荧光出生标记技术，用于在体内标记、追踪和分离小鼠胚胎中枢神经系统中的同型新生细胞群体。将羧基荧光素酯（CFSE）注入侧脑室可对有丝分裂（M 期）脑室区（VZ）祖细胞及其后代进行脉冲标记，这一过程我们称之为 FlashTag。M 期顶端祖细胞的特异性是由于这些细胞的体在细胞周期的这个阶段短暂接触脑室壁，使它们暴露于注入脑脊液的 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。标记的细胞可以在体外或固定组织中进行成像，用于电生理实验，或使用 FACS 进行细胞培养或（单细胞）RNA 测序分离。可以在 30 分钟内标记多个胚胎。染料保留数周，允许在产后识别标记的细胞。本方案描述了使用体内注射进行标记的程序、使用 FACS 分离活细胞的程序以及用于免疫组织化学的标记组织的处理程序。
