A Technique for Harvesting and Dissociating Dorsal Root Ganglia for Neuronal Cultures
Transcription
Take a section of a rat's spinal column and divide it to remove the spinal cord.
Detach the dorsal root ganglia, or DRGs, a cluster of neurons surrounded by satellite glial cells, or SGCs.
Place the DRGs in a growth medium. Remove the nerve roots to reduce SGC contamination.
Treat with collagenase to degrade the tissue matrix and wash to remove the enzymes.
Introduce trypsin to disrupt intercellular connections. Add a serum containing proteins that deactivate trypsin.
Add the growth medium and mechanically dissociate the tissue. Filter the suspension to isolate single cells and centrifuge to remove tissue debris.
Resuspend the cells to layer them on a density gradient medium, and centrifuge.
Neurons with a higher density settle at the bottom, facilitating the separation of SGCs.
Resuspend the neurons in a culture medium. Transfer them onto a culture substrate-coated well, allowing cell attachment.
Supplement with nerve growth factors for neuronal survival.
