Dorsal Root Ganglion Neuron Culture from Adult Rats

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