Dorsal Root Ganglion Neuron Culture from Neonatal Rats
Procedure
- Euthanize animal
- Remove spinal column from animal.
o You DO NOT need to remove spinal column under flow hood.
- Under hood, open spinal column to reveal spinal cord (open dorsal side).
o Make 2 cuts through top of spinal column (on either side). Remove a thin strip from top, about 1 mm wide. Make sure not to cut too far to sides; however you will need to remove enough bone to see DRG.
o You may wish to use a dissecting scope to check how much bone to remove.
- Remove spinal cord (optional). You will see DRG along either side of spinal column near the bottom.
- Under dissecting scope, remove DRG from spinal column & place into HBSS.
o DRG will be along either side of column, in small “pockets” in the bone. Each DRG will be round, with small pieces of nerve root on either side.
o To remove DRG from L4, L5 and L6, grasp DRG with micro forceps, then cut root on either side with micro scissors.
Plating DRG 1( Explant, non-dissociate DRG)
Intact DRGs are seeded in 24-well plate with 1ml of culture medium contains Neurobasal, B-27, L-glutamine, 5-FdU and Uridine. Medium will be changed every three days.
Plating DRG 2 (Dissocated DRG)
1) Incubate DRG in 0.125% collagenase for 90 minutes at 37 C°.
2) Remove the collagenase solution, add fresh collagenase, and incubate an additional 90 minutes at 37 C°
3) Remove the collagenase solution, and wash with HBSS by centrifugation at 600x g for 5 min.
4) Remove supernatant.
5) Resuspend cells in culture media, and triturate with 2 glass pipets (~ 15 x each): full diameter & ½ diameter (flame-narrowed).
6) Pass cells through cell-strainer into 50 ml-centrifuge tubes.
7) Resuspend cells in plating media & plate onto coated plates (about 5000 cells/cm2). Allow a few hours to adhere and add remainder of media.
8) Care of Cultures
- Exchange media every 3 days by changing 50% medium.
- Cells should survive up to two weeks with proper care; may be maintained longer if culture is healthy
Culture medium:
Neuralbasal media from Invitrogen
1x B-27
2 mM l-glutamine
17.5 μg/ml uridine and 7.5 μg/ml of 5-fluoro-2- deoxyuridine.
50 ng/ml NGF (OPTIONAL – mature DRG neuron don’t require to culture)
Rats
- Optimally use 3 to 5 days old rats; cells from older animals may be more difficult to maintain.