Abstract
The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.
Highlights
The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development[1,2]
We will describe procedures to obtain cultures of increasing purity[3,4], which can be used to study the differentiation of proliferating progenitor cells into granule neurons[5,6]
When large numbers of cells are needed for collecting protein or RNA samples, cells can be cultured on plastic culture dishes.The night before dissection, glass coverslips or plastic dishes for culture must be coated with poly-D-lysine (500 μg/ml; 800 μl/well for 6-well plates or 350 μl/well for 4-well plates). 5 mg/ml stock solution of poly-D-lysine is stored at -20°C, diluted in sterile distilled/deonized water and sterile filtered right before use
Summary
Glass coverslips (best if from Carolina Biological Supply Company) are washed with 10% HCl overnight at room temperature with mild agitation, rinsed thoroughly with distilled/deonized water and stored in 70% ethanol. Single coverslips are flame-sterilized by briefly holding them with forceps over an open flame (Bunsen burner) until the ethanol evaporates. 12-mm glass coverslips can be placed in 4-well culture plates and 25-mm coverslips in 6-well culture plates Single coverslips are flame-sterilized by briefly holding them with forceps over an open flame (Bunsen burner) until the ethanol evaporates. 12-mm glass coverslips can be placed in 4-well culture plates and 25-mm coverslips in 6-well culture plates
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