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BrainBits® Primary Dorsal Root Ganglion Culture Protocol
Enclosed are 10 pairs of E18 Sprague Dawley Rat dorsal root ganglion (DRG) in 2 ml of Hibernate® AB (HAB; Hibernate A®/B27®/GlutaMAX™; BrainBits® HAB 500 ml) and a 12 ml tube of NbActiv4® medium (Neurobasal®/B27®/GlutaMAX™; BrainBits® NbActiv4 500 ml) with 25 ng/ml Nerve Growth Factor (NGF). Use tissue immediately for highest cell yield, however, tissue can be stored for one week at 4-8oC.
Preparations (Room Temperature in a Sterile Hood):
- Prepare substrate by coating with poly-D-lysine (100 µg/ml) (Sigma P6407). Incubate coated surfaces for at least 1 hour (up to 20). Aspirate the poly-D-lysine, rinse once with ddH2O, aspirate and air dry.
- Prepare a 100 mg/ml stock solution of Collagenase/Dispase (Roche; Ref: 10 269 638 001) in ddH2O.
- To make the cell dissociation solution, dilute Step 2 to 1:100 with Hibernate A without Calcium and Magnesium (BrainBits® HA -Ca -Mg 500 mL) for a final working solution of 1 mg/ml collagenase (0.1 U) / dispase (0.8 U). Solution must be sterile filtered prior to use. Can be aliquoted and frozen @ -20ºC and thawed day of use.
- Fire polish the tip of a sterile 9” silanized pasteur pipette to an opening of ~0.5 mm (BrainBits® FPP).
- Aliquot 20 µl of Trypan Blue (Sigma: T8154) into a 0.5 ml tube for Step 9.
Cell Dispersal (Room Temperature in a Sterile Hood):
- With the silanized pasteur pipette, carefully remove the storage solution leaving the DRG’s in minimal solution. Save the storage solution in a sterile 15 ml tube for Step 4, below.
- Place tissue in cell dissociation solution (Step 3, above) for 1 hr and incubate at 37ºC (ambient atmosphere). Gently swirl every 5 min.
- Allow tissue to settle and remove cell dissociation solution leaving the tissue at the bottom in minimal solution.
- Immediately add 2 ml of Hibernate® AB (BrainBits® HAB; Hibernate A®/B27®/GlutaMAX™) from Step 1, above.
- With the silanized pasteur pipette, triturate tissue ~30 times until 90% dispered while avoiding air bubbles.
- Let undispersed pieces settle for 1 min.
- Transfer supernatant containing dispersed cells to a sterile 15 ml tube. Leave ~50 µl of HAB containing debris.
- Spin 1100 rpm (200 x G), 3 min. Discard supernatant leaving ~50 µl of HAB containing the pellet.
- Disperse the pellet of cells (flick the bottom of the tube with a finger) and resuspend pellet in 1 ml NbActiv4® with 25 ng/mL NGF.
- Aliquot 20 µl of cell solution into the 0.5 ml tube containing 20 µl of Trypan Blue (1:2 dilution).
- Count cells using a hemacytometer (calculate cells/ml).
Cell Plating (Room Temperature in a Sterile Hood):
- Dilute cells with NbActiv4® + 25 ng/ml NGF (0.2 ml/cm2) and plate at 100,000cells/cm2 or desired concentration.
- Incubate 37oC, 5% CO2, 9% O2, 95% humidity (or ambient O2).
- After 4 days, DRG’s display axons and dendrites; synapses and action potentials begin at 7 days.
- Change ½ of the medium with fresh, 37oC, CO2 equilibrated NbActiv4® + 25 ng/ml NGF every 3-4 days.
- Rinse twice with 37ºC HBSS (0.2 ml/cm2 of substrate).
- Prepare dye mix from an acetone stock of 15 mg/ml fluorescein diacetate and an aqueous stock of 4.6 mg/ml propidium iodide, dilute 15 µl of each into 1.5 ml HBSS (1:100 dilution).
- Add 20 µl of dye mix from step 2 to every 0.2 ml of HBSS added in step 1 (1:10 dilution).
- After ~1 min count live cells using blue excitation appropriate for fluorescein fluorescence (green cells). Count dead cells with green excitation for propidium iodide fluorescence (small red nuclei).
- Viability = (green cells/unit area)/(total cells plated/unit area) or Survival = green cells/(green + red cells)
For additional tips please see the FAQs at www.BrainBitsLLC.com.
Neurobasal®, B27®, GlutaMAX™, and Hibernate® are trademarks of Life Technologies.
Methods based on Brewer et al. (1993) J. Neurosci. Res. 35:567-576 (PMID: 8377226) and Brewer & Price (1996) Neuroreport 7:1509-1512 (PMID: 8856709).