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Nat Protoc. 2018 Oct;13(10):2297-2311. doi: 10.1038/s41596-018-0038-1.

In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag.

Author information

1
Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland.
2
Interfaculty Institute of Bioengineering, EPFL, Lausanne, Switzerland.
3
Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland. denis.jabaudon@unige.ch.
4
Clinic of Neurology, Geneva University Hospital, Geneva, Switzerland. denis.jabaudon@unige.ch.

Abstract

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.

PMID:
30258174
DOI:
10.1038/s41596-018-0038-1
[Indexed for MEDLINE]

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