Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture

1. Explant culture of DRG from Adult Mouse

1. Place the collected DRG in a 35 mm Petri dish containing 3 mL of ice-cold serum-free media (SFM).
2. Transfer each individual DRG in a dry glass Petri dish and, under a surgical microscope, clean and trim off excess fibers and connective tissue still attached to the DRG using a blade. The DRG is easily identifiable as a bulgy transparent structure along the white spinal nerve/root. Blood vessels often are found surrounding the DRG.
3. Place the cleaned DRG in a new Petri dish containing ice-cold SFM media.
4. Dilute the gelatinous protein mixture (see Table of Materials) in ice-cold SFM (1:1).
5. Plate the DRG ex vivo in 12-well plates pre-coated with 10/20 µL of gelatinous protein mixture and set them inside the incubator at 37 °C and 5% CO2 for 30-60 min.
6. Gently add 1.5-2 mL of SFM to the culture system to cover the entire explant and maintain the explants at culturing conditions (37 °C and 5% CO2).
   NOTE: This is a critical step because the DRG is anchored to the glass dishes only by the polymerized gelatinous protein mixture. Time of polymerization and pipetting skills are critical to avoid floating.
7. Change the medium of growing DRG every 72 hours and let the DRG grow for as long as needed.

2. Isolating Single Cell Neurons from DRG

1. Place all the DRG collected in a 1.5 mL sterile tube with 1.2 mL of F12 media containing 1.25 mg/mL of collagenase IV and incubate it at 37 °C and 5% CO2 for 45 min. Repeat this step for another 45 min after the first incubation.
2. Treat explants with 2 mL of F12 media containing trypsin (0.025%) for 30 min immediately after the collagenase IV treatment at 37 °C and 5% CO2.
3. Incubate with 2 mL of F12 media containing fetal bovine serum (FBS; 33%) at 37 °C and 5% CO2 for 15 min.
4. Wash explants three times with 2 mL of F12 media and proceed to mechanically dissociate them with a glass pipette until the media turns cloudy.
   NOTE: This procedure involves the collection of clean/trimmed DRG from the animal, as explained in the prior section. When performing mechanical dissociation of explants, be gentle and do not use excessive force since it can lead to spillage and loss of working material.
5. Filter the dissociated cell culture through a 0.22 µm filter to remove any impurities and excess connective tissue. Centrifuge the filtered cell lysate at (2,500 x g) for 2 min.
6. Remove the supernatant and resuspend the cell pellet in 500 µL of neurobasal media containing supplement for neuronal culture (1x), antibiotic mixture (1x), L-glutamate (0.5 mM), and nerve growth factor (5 µg/mL) (see Table of Materials).
7. Plate the dissociated cells onto laminin-coated cover slides (50 µg/mL; see Table of Materials) at a preferred cell density. Determine the cell density using a cell counter.
   NOTE: We plated cells at 25,000 cells/cover slide. This technique allows the dissociation of both neurons and glial satellite cells. The glial component co-cultured with the pseudounipolar neurons plays a critical role in neuronal survival.