DorsalRootGanglionNeuronCulturefromAdultRats

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