Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord
Transcription
Harvest mouse spinal cords and cut them into fragments.
Transfer the fragments to a tube containing a proteolytic enzyme and DNase. Vortex and incubate.
The enzyme digests the tissue's extracellular matrix, while DNase degrades contaminating free DNA.
Vortex again and pipette repeatedly to complete tissue dissociation, obtaining a single-cell suspension.
Centrifuge and remove the supernatant. Resuspend the cells in a solution containing DNase, and a protease inhibitor, and vortex. The inhibitor neutralizes the enzyme.
Layer fresh inhibitor solution on the top, centrifuge, and remove the supernatant.
Resuspend the cells in density gradient media, vortex, and then centrifuge.
The cells settle at the bottom, while the myelin debris remains at the top.
Discard the debris. Wash repeatedly with buffer, centrifuge, and remove the supernatant. Resuspend the cells in growth media.
Plate the cells. The uncoated surface facilitates glial cell attachment.
Remove the spent media and add fresh media to induce glial cell proliferation, establishing a mixed glial culture.
