Establishing a Primary Dorsal Root Ganglia Cell Culture from a Rat Spinal Column
Transcription
Take a rat spinal column. Make two cuts along its sides and one lateral cut.
Remove the dorsal muscles and the dorsal portion of the vertebrae.
Extract the spinal cord.
Identify the dorsal root ganglia, or DRGs, located bilaterally along the lumbar vertebrae.
The DRGs contain neurons surrounded by glial cells.
Excise the DRGs and collect them in a dish. Wash the tissue repeatedly with serum-free media.
Transfer the DRGs to a plate containing collagenase solution and incubate. Collagenase degrades the extracellular matrix.
Wash with buffer. Add trypsin-EDTA and incubate to disrupt cell-cell junctions, loosening the cells.
Transfer the DRGs to a tube, centrifuge, and discard the supernatant.
Resuspend the tissue in culture media and pipette repeatedly to release the cells.
Transfer the cells onto a polymer-coated culture plate well to facilitate attachment.
Remove the media and add fresh media containing inhibitors and growth factors.
The inhibitors limit glial cell proliferation, while the growth factors promote neuronal growth.
