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Isolation and culture of adult neurons and neurospheres

Nature Protocols volume 2, pages 1490–1498 (2007)Cite this article

Abstract

Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1–3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.

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Figure 1
Figure 2: OptiPrep gradient separation of cortical neurons pooled from the brains of two 6-month-old C57/BL6 mice.
Figure 3: Hippocampal neurons in culture after isolation from a 6-month-old C57/BL6 mouse.
Figure 4: Cortical neurospheres from a 6-month-old C57/BL6 mouse.

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Acknowledgements

This work was supported by a Temple Foundation award from the Alzheimer Association and the National Institute on Aging.

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  1. Department of Neurology and Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, 62794-9626, Illinois, USA

    Gregory J Brewer & John R Torricelli

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  1. Gregory J Brewer
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Correspondence to Gregory J Brewer.

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Competing interests

Southern Illinois University receives royalties from Invitrogen for Neurobasal™ and B27™ products. In partnership with his wife, Brewer is the founder and owner of BrainBits LLC, maker of Hibernate™.

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Brewer, G., Torricelli, J. Isolation and culture of adult neurons and neurospheres. Nat Protoc 2, 1490–1498 (2007). https://doi.org/10.1038/nprot.2007.207

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