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

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

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.

MeSH terms

  • Animals
  • Batch Cell Culture Techniques / methods*
  • Brain / cytology*
  • Brain / metabolism*
  • Cell Culture Techniques / methods
  • Cell Separation / methods*
  • Cells, Cultured
  • Female
  • Flow Cytometry / methods*
  • Gene Expression Profiling / methods
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Neurons / cytology*
  • Neurons / metabolism*