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J Vis Exp. 2011 Sep 17;(55). pii: 3196. doi: 10.3791/3196.

Preparation of synaptoneurosomes from mouse cortex using a discontinuous percoll-sucrose density gradient.

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  • 1Department of Pathology and Laboratory Medicine, Waisman Center for Developmental Disabilities, University of Wisconsin, USA.

Abstract

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters. The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

PMID:
21946781
[PubMed - indexed for MEDLINE]
PMCID:
PMC3230173
Free PMC Article
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