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


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.


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.


CONCLUSIONS
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.
Institute for Molecular Medicine, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
Sabina
S
Berl
Institute for Molecular Medicine, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
Khalad
K
Karram
Center for Integrative Physiology and Molecular Medicine (CIPMM), Molecular Physiology, University of Saarland, Building 48, D-66421 Homburg, Germany.
Anja
A
Scheller
Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, D-51429 Bergisch-Gladbach, Germany.
Melanie
M
Jungblut
Center for Integrative Physiology and Molecular Medicine (CIPMM), Molecular Physiology, University of Saarland, Building 48, D-66421 Homburg, Germany.
Frank
F
Kirchhoff
Institute for Molecular Medicine, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. Electronic address: waisman@uni-mainz.de.
Ari
A
Waisman
D000818:Animals; D061249:Batch Cell Culture Techniques / Q000379:methods*; D001921:Brain / Q000166:cytology* / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*
Adult mice; Enrichment; Ex vivo; FACS sorting; Isolation; Magnetic-beads; Neurons; RNA isolation