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

Abstract

BackgroundIsolation 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 methodsHere 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. ResultsEnrichment 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 methodsFormer 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. ConclusionThese 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.