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J Vis Exp. 2015 Sep 14;(103). doi: 10.3791/53247.

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics.

Author information

1
CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967; INSERM, UMR 967; Université Paris Diderot, Sorbonne Paris Cité, UMR 967; Université Paris Sud, UMR 967; CNRS, Université Paris Sud, UMR 9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department; mathieu.daynac@cea.fr.
2
CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967; INSERM, UMR 967; Université Paris Diderot, Sorbonne Paris Cité, UMR 967; Université Paris Sud, UMR 967; lise.morizur@cea.fr.
3
CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967; INSERM, UMR 967; Université Paris Diderot, Sorbonne Paris Cité, UMR 967; Université Paris Sud, UMR 967.
4
CNRS, Université Paris Sud, UMR 9197, Neuroscience Paris-Saclay Institute, Molecules Circuits Department.
5
CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967; INSERM, UMR 967; Université Paris Diderot, Sorbonne Paris Cité, UMR 967; Université Paris Sud, UMR 967; marc-andre.mouthon@cea.fr.
6
CEA DSV iRCM SCSR, Laboratoire de Radiopathologie, UMR 967; INSERM, UMR 967; Université Paris Diderot, Sorbonne Paris Cité, UMR 967; Université Paris Sud, UMR 967; boussin@cea.fr.

Abstract

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

PMID:
26436641
PMCID:
PMC4692602
DOI:
10.3791/53247
[Indexed for MEDLINE]
Free PMC Article

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