Generating Dorsal Root Ganglion Explant and Dissociated Cell Culture

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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.

Материалы

Name	Company	Catalog Number	Comments
Adult Mice NIH/Swiss	Harlan Laboratories
35mm petri dish	Cell Treat	229635
Matrigel ECM	Sigma-Aldrich	E1270	Gelatinous protein mixture
F12 Media	Gibco	11765-054	*Part of serum-free media
Collagenase IV	Sigma-Aldrich	C5138
Trypsin	Sigma-Aldrich	25200-056
FBS	Sigma-Aldrich	F6178
0.22um filter	BD Falcon	352350
Neurobasal media	Gibco	10888-022
B27 supplement	Gibco	17504-044	Supplement for neuronal culture
PSN antibiotics	Gibco	15640-055	*Part of serum-free media Antibiotic mixture
L-glutamate	Sigma-Aldrich	G7513	*Part of serum-free media
NGF	Alomone Labs	N-100	Nerve growth factor
Laminin coated coverslide	Neuvitro	GG-14-Laminin
ONPG subtrate	Pierce	34055
X-gal	Invitrogen	15520034
BSA	Sigma-Aldrich	A2153-100G	*Part of serum-free media
BME	Gibco	21010-046	*Part of serum-free media
Glucose	Sigma-Aldrich	G7021-1KG	*Part of serum-free media
KIT (Insulin-transferrin-Selenium-A)	Gibco	51300-044	*Part of serum-free media
Vitamin-C	Sigma-Aldrich	A4403	*Part of serum-free media
Putrescine	Sigma-Aldrich	P7505	*Part of serum-free media
PBS	Gibco	10010-031
Triton-X	Sigma-Aldrich	T9284-500ML