A Technique to Culture Mouse Hippocampal Neurons
Trascrizione
Take a harvested mouse pup hippocampus in a buffer.
Cut the hippocampus into smaller fragments and transfer them to a conical tube.
Wash the tissue with buffer. Once the fragments settle, remove the buffer. Now, add fresh buffer and trypsin.
During incubation, trypsin, a proteolytic enzyme, digests the tissue's extracellular matrix, loosening the hippocampal neurons.
Wash the digested tissue with buffer.
Once the fragments settle, remove the buffer. Add fresh buffer and pipette the tissue repeatedly to mechanically dissociate it, obtaining a single-cell suspension.
Once the undigested tissue settles, transfer the cell suspension onto poly-L-lysine-coated coverslips with wax supports within a culture plate containing media.
The cells attach to the poly-L-lysine-coated surfaces through electrostatic interactions.
Now, place the neuron-adhered coverslips facing down into a dish containing cortical glial or non-neuronal cells in serum-free media.
In culture, wax supports prevent direct contact between neurons and glial cells, creating a microenvironment where glial-cell-secreted factors promote neuronal survival and maturation.
