Isolation and Culture of Mouse Primary Cerebellar Granule Neurons
Transcription
Take a freshly harvested mouse pup brain.
Separate the cerebellum and remove its membranous layer.
From the ventral side, remove the network of blood vessels.
Chop the cerebellum into fragments and transfer them to a tube containing buffer.
Centrifuge and discard the supernatant containing debris.
Add trypsin, a proteolytic enzyme, to digest the tissue's extracellular matrix and loosen the cells.
Add a buffer containing trypsin inhibitor and DNase.
The inhibitor stops trypsin activity while DNase degrades any contaminating DNA.
Mechanically dissociate the tissue to form a cell suspension, including, cerebellar granule neurons and non-neuronal or glial cells.
Transfer the cell suspension to a tube.
Add buffer. Centrifuge and remove the supernatant. Resuspend cells in media and plate them onto culture plates coated with poly-D-lysine.
The cells attach to the coated surfaces.
Add an antimetabolite to eliminate proliferating glial cells and generate a pure culture of cerebellar granule neurons.
