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J Neurosci Methods. 2017 May 1;283:15-22. doi: 10.1016/j.jneumeth.2017.03.015. Epub 2017 Mar 21.

Enrichment and isolation of neurons from adult mouse brain for ex vivo analysis.

Author information

1
Institute for Molecular Medicine, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
2
Center for Integrative Physiology and Molecular Medicine (CIPMM), Molecular Physiology, University of Saarland, Building 48, D-66421 Homburg, Germany.
3
Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, D-51429 Bergisch-Gladbach, Germany.
4
Institute for Molecular Medicine, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. Electronic address: waisman@uni-mainz.de.

Abstract

BACKGROUND:

Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases.

NEW METHODS:

Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads.

RESULTS:

Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation.

COMPARISON WITH EXISTING METHODS:

Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression.

CONCLUSION:

These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS.

KEYWORDS:

Adult mice; Enrichment; Ex vivo; FACS sorting; Isolation; Magnetic-beads; Neurons; RNA isolation

PMID:
28336359
DOI:
10.1016/j.jneumeth.2017.03.015
[Indexed for MEDLINE]

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