Protocols

Dorsal Root Ganglion Neuron Culture from Adult Rats

Posted on Sunday, June 12, 2005

Description
Procedure for culturing and maintaining Dorsal Root Ganglion (DRG) neurons from adult rats.

Procedure
Preparing Culture Surface
- Collagen coat cover slips (if using) or cell culture dishes: evenly spread 2-3 drops of rat-tail collagen onto culture surface and allow to dry. May store overnight in cell culture hood or up to 7 days at 37C.
Removing DRG
- 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 2.5 % collagenase.
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, grasp DRG with micro forceps, then cut root on either side with micro scissors.
Plating DRG
- Incubate DRG in 2.5% collagenase for an additional 20 minutes at 37 C.
- Add calf serum & spin DRG for 5 minutes (1000 RPM). Remove supernatant.
- Incubate in 0.5% trypsin for 15 minutes at 37 C.
- Add calf serum & spin DRG for 5 minutes (1000 RPM). Remove supernatant.
- Resuspend cells in plating media, and triturate with 2 calf serum-coated glass pipets (~ 15 x each): full diameter & ½ diameter (flame-narrowed).
- Pass cells through cell-strainer into 50 ml-centrifuge tubes
- Resuspend cells in plating media & plate onto collagen-coated cover slips (about 10-drops per well). Allow a few hours to adhere and add remainder of media.
Care of Cultures
- Exchange media every 48 hours.
- Cells should survive up to two weeks with proper care; may be maintained longer if culture is healthy


Recipes
Plating/Feeding Media
* Replace media every 48 hours
Neuralbasal-A media with:
1x B-27 WITH antioxidants
1x Pen-strep-neo
0.23 mM l-glutamine
FUDR (OPTIONAL – for cultures without Schwann Cells)
10 ng/ml NGF (OPTIONAL – adults don’t require to culture)


Supplies
Rats
- Optimally use 12 week old male rats; cells from older animals may be more difficult to maintain.
- One 12 week old adult male will yield enough cells for about 6-12 wells (using 12-well plates). You may wish to use several animals to ensure enough cells.
Surgical Tools
* Use sterile tools for all steps
- To remove spinal column:
o Scissors
o Forceps
o Bone cutters
- To open spinal column:
o Bone cutters (RS8480), small
o Large bone cutters or Rangeurs
o Scissors
o Small forceps (NOT micro forceps)
- To remove DRG:
o Micro scissors
o Micro forceps
Reagents
- Euthanasia Drugs (10:3 Ketamine-Rompun or Phenobarbital)
- Rat-tail collagen
- Calf serum
- 2.5% collagenase
- 0.5% trypsin
- Neuralbasal-A media (Gibco)
- B-27 with antioxidants
- L-glutamine
- Pen-strep-neo
- OPTIONAL: NGF, FUDR
Miscellaneous
- Glass cover slips (OPTIONAL - preferably German glass, sized to fit wells of cell culture dish).
- Cell culture dishes (6- or 12-well)
- Glass pipets
- Cell strainer (BD Falcon 70 M nylon #352350)
- Sterile centrifuge tubes – 50 ml


Tips


Submitted by: alicat