Abstract
Primary neurons from rodent brain hippocampus and cortex have served as important tools in biomedical research over the years. However, protocols for the preparation of primary neurons vary, which often lead to conflicting results. This report provides a robust and reliable protocol for the production of primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-neuronal cells. The neurons were grown in serum-free media and maintained for several weeks without any additional feeder cells. The neuronal cultures maintained according to this protocol differentiate and by 3 weeks develop extensive axonal and dendritic branching. The cultures produced by this method show excellent reproducibility and can be used for histological, molecular and biochemical methods.
Highlights
A primary neuron culture from embryonic rodent hippocampus or cortex has been one of the most fundamental methodologies for modern neurobiology
All embryos staged at embryonic day 17 (E17)–18 from the female rats were used in the experiments
Trituration of cortical neurons is harsher, and the cells need to be stained with Trypan Blue to exclude dead cells
Summary
A primary neuron culture from embryonic rodent hippocampus or cortex has been one of the most fundamental methodologies for modern neurobiology. By modifying the culture medium and conditions, numerous factors responsible for directing different aspects of neuronal survival, differentiation and phenotype have been revealed [1]. Earlier publications have more or less thoroughly described protocols for cultures of primary neurons over the years. Original methods have used serum to support neuronal survival and differentiation [2] but more recently culture methods using defined media without serum have been introduced [3,4,5,6]. Glial cells provide critical support to cultured neurons [7,8,9]. Several methods to keep excess glial proliferation in check or to prevent mixing between neurons and glia have been described [10]
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