This work was supported by a Project Grant from the Canadian Institutes of Health Research (FRN-162434) and a Discovery Grant from the MS Society of Canada (EGID-3761). The authors would like to thank Dr. Sun and the Cross Cancer Institute for training and use of the ImageXpress system.

All animal experiments were performed with approval from the University of Alberta Health Sciences Animal Care and Use Committee (protocol 0000274).
1. Reagent preparation
<ol>
	<li>For day 1, prepare the following reagents: cell culture grade water; poly-D-lysine-prepare a stock by reconstituting the whole bottle in 50 mL of culture grade water to a stock concentration of 100 &#181;g/mL, store at 4 &#176;C, and dilute the stock 1:10 in culture grade water to a final concentration of 10 &#181;g/mL; tris-HCl-prepare a stock by dissolving powdered tris hydrochloride in culture grade water to a stock concentration of 500 mM (3.94 g in 50 mL), pH 9.5, filter sterilize (0.22 &#181;m), store at 4 &#176;C, and dilute the stock 1:10 in culture grade water to a final concentration of 50 mM; secondary antibodies (goat anti-rat, -rabbit, and -mouse).</li>
	<li>For day 2, prepare the following reagents.
	<ol>
		<li>Prepare cell culture grade Ca2+, Mg2+-free Hank&#39;s balanced salt solution (HBSS; HBSS-/-), and cell culture grade D-phosphate buffered saline (PBS) with Ca2+ and Mg2+. Prepare a laminin stock aliquot and store at -80 &#176;C immediately upon receiving; dilute the stock in sterile HBSS-/- to a final concentration of 10 &#181;g/mL for working stock.</li>
		<li>Prepare 4% bovine serum albumin (BSA) stock by dissolving 400 mg of BSA in 7.5 mL of D-PBS (pH 7.4), bring the volume to 10 mL, filter sterilize (0.22 &#181;m), aliquot, and store at -20 &#176;C. Prepare 0.2% BSA by diluting 750 &#181;L of 4% BSA in 14.25 mL of D-PBS.</li>
		<li>Prepare 0.4% DNAse stock by adding 1 mL of Earle&#39;s balanced salt solution (EBSS) per 12,500 units of DNAse, filter sterilize (0.22 &#181;m), aliquot, and store at -20 &#176;C. Prepare panning buffer (0.02% BSA) by adding 3 mL of 0.2% BSA and 100 &#181;L of 0.4% DNAse in 27 mL of D-PBS. Prepare primary antibodies (CD45, PDGRF&#946;, O4). 
		NOTE: This protocol uses an O4 hybridoma8,9, however, 10 &#181;g of a commercially available O4 antibody would also be appropriate.</li>
		<li>Prepare the combination collagenase stock by dissolving a bottle of combination collagenase (50 mg) in 5 mL of HBSS-/-, aliquot, and store at -20 &#176;C. Prepare a working stock per two mice by adding 500 &#181;L of warmed combination collagenase and 50 &#181;L of warmed 0.4% DNase to 4.5 mL of HBSS-/-.</li>
		<li>Prepare low ovomucoid by adding 3 g of BSA to 150 mL of D-PBS, then add 3 g of trypsin inhibitor and mix to dissolve. Adjust the pH to 7.4 and bring the volume up to 200 mL with D-PBS. Filter sterilize, prepare 1 mL aliquots, and store at -20 &#176;C. On the day of dissection, combine 1 mL of low ovomucoid in 9 mL of D-PBS for the working solution.</li>
		<li>Prepare 15% BSA by dissolving 7.5 g of BSA in 50 mL of HBSS-/- (place on a shaker to dissolve fully), filter sterilize, and store at 4 &#176;C.</li>
		<li>Prepare 50 mL of neuron media by adding 23.2 mL of DMEM, 23.2 mL of neurobasal medium, 500 &#181;L of glutamax, 500 &#181;L of sodium pyruvate, 500 &#181;L of penicillin/streptomycin (aliquot immediately upon arrival and store at -20 &#176;C) , 500 &#181;L of insulin (5 &#181;g/mL final), 500 &#181;L of SATO, 50 &#181;L of N-acetyl-L-cysteine (NAC; 5 &#181;g/mL final), and 1,000 &#181;L of B27+ (immediately aliquot and store at -20 &#176;C upon receiving).
		<ol>
			<li>Prepare the insulin stock by dissolving in culture grade water to a concentration of 0.5 mg/mL (50 mg in 100 mL), adjust the pH to 7.4, filter sterilize (0.22 &#181;m), prepare 500 &#181;L aliquots, and store at -20 &#176;C.</li>
			<li>Prepare the SATO stock by combining 20 &#181;L of progesterone (2.5 mg in 100 &#181;L of ethanol), 800 &#181;L of sodium selenite (4 mg in 10 mL of neurobasal media + 10 &#181;L of 1 N NaOH), 800 mg of BSA, 800 mg of transferrin, 128 mg of putrescine dihydrochloride, and 80 mL of neurobasal. Filter sterilize (0.22 &#181;m), prepare 500 &#181;L aliquots, and store at -20 &#176;C. It is essential to make fresh progesterone and sodium selenite solutions.</li>
			<li>Prepare 50 &#181;L of N-acetyl-L-cysteine (NAC) stock by dissolving in neurobasal medium to a stock concentration of 5 mg/mL (50 mg in 10 mL), filter sterilize (0.22 &#181;m), aliquot, and store at -20 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>For day 4 and 5, prepare the following reagents.
	<ol>
		<li>Prepare 0.2 M phosphate buffer (PB) by dissolving 21.8 g of sodium phosphate dibasic (Na2HPO4) and 8.34 g of sodium phosphate monobasic dihydrate (NaH2PO4) in 1,000 mL of water. Adjust the pH to 7.4 and store at room temperature.</li>
		<li>Prepare 8% paraformaldehyde (PFA) as follows: in a fume hood, warm 50 mL of water to 55 &#176;C, turn off the heat, and add 8 g of PFA prills, stirring constantly. Add 1 N NaOH dropwise until the solution begins to clear, then add 40 mL of 0.2 M PB, and continue stirring until clear. Cool the solution to room temperature, adjust the pH to 7.4, and bring the volume up to 100 mL with 0.2 M PB. Filter and store at 4 &#176;C for up to 2 weeks.</li>
		<li>Prepare D-PBS + Triton X-100 (PBSTX) by adding 0.2% Triton X-100 in culture grade D-PBS (1 mL of Triton + 500 mL of D-PBS). Store at 4 &#176;C. Prepare blocking solution, which is 1% normal donkey serum (NDS) in PBSTX. Ahead of time, 1 mL of NDS + 9 mL of PBSTX can be prepared and stored at -20 &#176;C in 10 mL aliquots.</li>
		<li>Prepare antibody solution which is 0.2% NDS/0.2% BSA in PBSTX : 200 &#181;L of NDS + 200 mg BSA + 9.6 mL of PBSTX; can be prepared ahead of time and stored at -20 &#176;C in 10 mL aliquots.</li>
	</ol>
	</li>
</ol>
2. Equipment setup
<ol>
	<li>Set up the following equipment: sterile cell culture biosafety cabinet, micropipette set, serological pipettes and pipette gun, 10 cm Petri dishes, desired cell culture dishes for plating (here, 24-well black glass-bottom plates were used for imaging), 15 mL and 50 mL conical tubes, dissection tools (namely: fine spring scissors for laminectomy and nerve trimming, fine forceps, and a razor blade), water bath set to 37 &#176;C, 70 &#181;m cell strainer, centrifuge (10 min at 300 x g) with adaptors for 15 mL conical tubes, trypan blue and haemocytometer, fume hood, and magnetic stirring hot plate.</li>
</ol>
3. Experimental method
<ol>
	<li>Day 1: preparation of dishes
	<ol>
		<li>In a biosafety cabinet, coat the desired culture surface with enough poly-D-lysine to cover the bottom of the well. Store overnight at 4 &#176;C. Here, 24-well glass-bottom plates were used for imaging, and thus 300 &#181;L of poly-D-lysine was used.</li>
		<li>In a biosafety cabinet, prepare the panning dishes: for the CD45 dish, add 10 mL of working tris-HCl solution and 30 &#181;L of goat anti-rat IgG to a 10 cm Petri dish; for the PDGFR&#946; dish, add 10 mL of working tris-HCl solution and 30 &#181;L of goat anti-rabbit IgG to a 10 cm Petri dish; for the O4 hybridoma dish, add 10 mL of working tris-HCl solution and 30 &#181;L of goat anti-mouse IgG to a 10 cm Petri dish. Ensure the solution covers the entire dish area and leave at 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Day 2: dissection, trituration, and immunopanning
	<ol>
		<li>Aspirate the poly-D-lysine, wash the plates three times with tissue culture water, tilt the lid open, and allow the surface to dry completely in a biosafety cabinet. Apply enough laminin to the plates to coat the bottom of each well, and place in an incubator at 37 &#176;C. For the 24-well plates, 200 &#181;L is sufficient.</li>
		<li>Finish preparing the panning dishes. Wash each dish with D-PBS and incubate with 10 &#181;g of primary antibody. Allow to sit at room temperature for at least 2 h.</li>
		<li>For the CD45 dish, add 5 mL of 0.2% BSA and 20 &#181;L of CD45 primary; for the PDGFR&#946; dish, add 5 mL of 0.2% BSA and 15.4 &#181;L of PDGFR&#946;; for the O4 dish, add 3 mL of 0.2% BSA, 2 mL of O4 hybridoma, and an additional 100 &#181;L of 4% BSA (to ensure the final BSA concentration remains at 0.2%).</li>
		<li>Euthanize one to four mice using 0.1 mL/kg sodium pentobarbitol (per 10 cm immunopanning dish). We used C57BL/6 mice at 8-10 weeks old. Both sexes were used and cultured separately from one another. Perfuse the animal with 30 mL of cold 0.9% saline.</li>
		<li>Pin the front and hind paws to a styrofoam stage and use a clean razor blade to expose the spinal column from the back. Perform a laminectomy by making cuts about half-way deep on either side of the dorsal spinal column, removing the dorsal half of the column, to expose the spinal cord. Either gently cut the nerves and remove the spinal cord, or gently push the cord to the side, maintaining the nerve integrity.</li>
		<li>Use the spinal nerve roots (either severed or attached to the spinal cord) to find and remove all the DRGs from either side of the spinal column. Trim the nerve debris from each DRG as it is removed (more nerve debris in the culture may lead to poor culture survival). Collect the DRGs in 15 mL of HBSS-/- in a 15 mL conical tube on ice. 
		NOTE: Note that the tissue is dissected in open air, but once the DRGs are added to the tube they should be treated as sterile and only manipulated in a biosafety cabinet.</li>
		<li>Place frozen stocks of stemxyme 1 and 0.4% DNAse in a water bath, approximately 30 min prior to the end of the dissections (roughly when starting the last mouse). Allow them to settle to the bottom of the conical tube, then continue the protocol in a biosafety cabinet.</li>
		<li>Aspirate most of the HBSS-/- from the DRGs. Use a pipette rather than suction to get &#60;1 mL of liquid, and leave ~100 &#181;L liquid so as not to risk losing tissue.</li>
		<li>Wash three times with 1 mL of HBSS-/-. Add 5 mL of working stemxyme solution to the tissue. Cover the lid with a transparent film and float the tube on its side in a water bath set to 37 &#176;C for 1 h.</li>
		<li>After incubation in the enzyme, add 1 mL of low ovo inhibitor solution to the tube. Triturate the cells gently with a p1000 pipette 10-15 times. Allow the tissue chunks to settle.</li>
		<li>Transfer the top 2-3 mL of solution (containing dissociated cells) to fresh low ovomucoid solution. Repeat until the tissue is fully dissociated and no visible chunks remain.</li>
		<li>Centrifuge for 10 min at 300 x g at room temperature. Siphon off the supernatant again, using a pipette rather than suction for the last 1 mL, leaving ~100 &#181;L, and resuspend the cell pellet in 1 mL of panning buffer.</li>
		<li>Gently pipette to mix. Pre-wet a 70 &#181;m cell strainer with 1 mL of panning buffer over a 50 mL conical tube. Filter the cell solution through the cell strainer.</li>
		<li>Wash the tube with 1 mL of panning buffer, then pass through the strainer (3 mL of filtrate). Layer the cell suspension gently (1 mL at a time) on top of 2 mL of a 15% BSA cushion to remove myelin debris. A 2 mL cushion is effective for two mice, and 3 mL is effective for four mice.
		<ol>
			<li>For layering, place the 2 mL of BSA in the tube, close, and gently coat the sides of the tube with BSA. Then, gently layer the cell suspension of top, by pipetting against the side of the tube.</li>
		</ol>
		</li>
		<li>Centrifuge at room temperature at 300 x g for 10 min (slow acceleration and deceleration). Use a P1000 pipette to remove the clear liquid on top, the myelin phase in between, and the BSA, 1 mL at a time (changing tips in between each mL). Leave ~100 &#181;L of BSA so as not to disturb the pellet.</li>
		<li>Add 5 mL of panning buffer and incubate the tube in a 37 &#176;C, 10% CO2 incubator for 30-45 min. This allows for antigen retrieval. Be sure to loosen the cap to allow gas exchange.</li>
		<li>Wash the CD45 dish three times with D-PBS. Pour off the final rinse. Decant the cell suspension into the CD45 dish.</li>
		<li>Incubate the cells on the CD45 dish for a total of 20 min at room temperature, swirling gently at the 10 min point to allow the cells equal access to the antibody.</li>
		<li>Rinse the PDGFR&#946; dish three times with D-PBS and pour off the final rinse. Transfer the unbound cells from the CD45 dish to the PDGFR&#946; dish.</li>
		<li>Gently shake the CD45 dish and prop it up at an angle. Gently pipette 1 mL of the cell suspension over the dish to collect unbound cells. Decant it into the PDGFR&#946; dish and pipette to transfer the remaining cell suspension to the PDGFR&#946; plate.</li>
		<li>Incubate the PDGFR&#946; plate for a total of 20 min at room temperature, swirling gently at the 10 min point to allow the cells equal access to the antibody.</li>
		<li>Rinse the O4 dish three times with D-PBS and pour off the final rinse. Transfer the unbound cells from the PDGFR&#946; dish to the O4 dish using the same method as in step 3.2.19. Incubate the O4 plate for a total of 20 min at room temperature, swirling gently at the 10 min point to allow the cells equal access to the antibody.</li>
		<li>Transfer the cell suspension to a 15 mL conical tube and centrifuge for 10 min at 300 x g. Resuspend the cells in the desired volume, and dilute 1:1 with trypan blue for cell viability. Count medium to large cells with a haemocytometer (this will allow for the best representation of the number of neurons in the culture).</li>
		<li>Remove the laminin and plate the cells at the desired density. Do not allow plate to dry between laminin removal and plating, and add the cells immediately.</li>
		<li>Culture the quantified cells for neuronal enrichment (Figure 3) with 500 neurons in 25 &#181;L of culture medium (described in 1.2.7) per well in 24-well plates and incubate at 37 &#176;C for 30 min. Flood the wells to a final volume of 500 &#181;L.</li>
	</ol>
	</li>
	<li>Day 4: immunocytochemistry (ICC) fixation, blocking, and primary
	<ol>
		<li>After 48 h of growth, add 500 &#181;L/well (or volume equivalent to the media in the well) of 8% PFA for 15 min at room temperature to fix the cells. If media analysis is desired, one may fix with 4% PFA directly. However, we have not tested this.</li>
		<li>Wash the cells with D-PBS three times. Note, we have kept these cells in culture without media change for a maximum of 72 h, but hypothesize they could survive longer, particularly if the media is half-changed out.</li>
		<li>Remove the final wash and add enough blocking solution per well to cover the cells completely. Block for 30 min at room temperature. Remove the blocking solution and add primary antibody in antibody solution: &#946;3-tubulin at a 1:1,000 dilution. Seal the plate with transparent film and incubate overnight at 4 &#176;C. 
		NOTE: One might consider staining with PDGFR&#946; for the absence of fibroblasts, CD45 for the absence of immune cells, P0 or PMP22 for the absence of myelin, or fabp7 for satellite glia.</li>
	</ol>
	</li>
	<li>Day 5: ICC secondary
	<ol>
		<li>Wash the cells with D-PBS three times. Remove the final wash and add secondary antibodies in antibody solution: anti-rabbit 488 at a 1:500 dilution and DAPI at a 1:2,000 dilution. Incubate for 30 min at room temperature, protected from light.</li>
		<li>Wash with D-PBS three times. Add enough D-PBS to cover the bottom of the well, and image. The plate can be sealed with transparent film, protected from light, and stored at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Haberberger, R. V., Barry, C., Dominguez, N., Matusica, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dorsal+root+ganglia.">Human dorsal root ganglia.</a> Frontiers in Cellular Neuroscience. 13, 271 (2019).</li><li>Perner, C., Sokol, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dissection+and+culture+of+murine+dorsal+root+ganglia+neurons+to+study+neuropeptide+release.">Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release.</a> STAR Protocols. 2 (1), 100333 (2021).</li><li>Lin, Y. T., Chen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+root+ganglia+isolation+and+primary+culture+to+study+neurotransmitter+release.">Dorsal root ganglia isolation and primary culture to study neurotransmitter release.</a> Journal of Visualized Experiments:JoVE. (140), e57569 (2018).</li><li>Foo, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+method+for+the+purification+and+culture+of+rodent+astrocytes.">Development of a method for the purification and culture of rodent astrocytes.</a> Neuron. 71 (5), 799-811 (2011).</li><li>Nolle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+Glial+Cells+From+Human+Post-mortem+Tissue+for+Transcriptome+and+Proteome+Analysis+Using+Immunopanning.">Enrichment of Glial Cells From Human Post-mortem Tissue for Transcriptome and Proteome Analysis Using Immunopanning.</a> Frontiers in Cellular Neuroscience. 15, 772011 (2021).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+and+troubleshooting+immunopanning+protocols+for+purifying+neural+cells.">Designing and troubleshooting immunopanning protocols for purifying neural cells.</a> Cold Spring Harbor Protocols. 2014 (12), 1342-1347 (2014).</li><li>Zuchero, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+dorsal+root+ganglion+neurons+from+rat+by+immunopanning.">Purification of dorsal root ganglion neurons from rat by immunopanning.</a> Cold Spring Harbor Protocols. 2014 (8), 826-838 (2014).</li><li>Sommer, I., Schachner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+(O1+to+O4)+to+oligodendrocyte+cell+surfaces:+an+immunocytological+study+in+the+central+nervous+system.">Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system.</a> Developmental Biology. 83 (2), 311-327 (1981).</li><li>Sommer, I., Schachner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+that+are+O4+antigen-positive+and+O1+antigen-negative+differentiate+into+O1+antigen-positive+oligodendrocytes.">Cell that are O4 antigen-positive and O1 antigen-negative differentiate into O1 antigen-positive oligodendrocytes.</a> Neuroscience Letters. 29 (2), 183-188 (1982).</li><li>Hanani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+glial+cells+in+sensory+ganglia:+from+form+to+function.">Satellite glial cells in sensory ganglia: from form to function.</a> Brain Research. Brain Research Reviews. 48 (3), 457-476 (2005).</li><li>Viveiros, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-cell+labeling+coupled+to+direct+analysis+of+extracellular+vesicles+in+the+conditioned+medium+to+study+extracellular+vesicles+secretion+with+minimum+sample+processing+and+particle+loss.">In-cell labeling coupled to direct analysis of extracellular vesicles in the conditioned medium to study extracellular vesicles secretion with minimum sample processing and particle loss.</a> Cells. 11 (3), 351 (2022).</li></ol>

The authors have no conflicts of interest to declare.

This immunopanning protocol increases the proportion of neuronal cells in DRG primary cultures. For the best results, dissections should be done in a timely manner, and DRGs should be trimmed of excess nerves. The dissociation step should be carefully monitored, and should not exceed 1 h, to prevent the cells from unnecessary stress. With regard to the immunopanning specifically, each plate should be gently swirled at the halfway point to allow the cells to have access to the coated antibodies. The plates should also be ...

Dorsal root ganglia (DRGs) house the cell bodies of the sensory neurons which innervate peripheral tissues. Studying these neurons allows us to understand the mechanistic underpinnings of pain and sensory conditions. However, DRGs are not comprised of neurons alone, but also contain fibroblasts, Schwann cells, macrophages, and other immune cells1. Despite the presence of these various cell types, whole DRG cultures are often referred to in the literature as neuronal2,3. These cultures are still useful for neuronal investigation by imaging or flow cytometry, which would allow for neuronal identification by staining, cell size, and/or morphology. However, for assays such as polymerase chain reaction (PCR) or western blotting, where cultures are homogenized for RNA or protein collection, the presence of non-neuronal cell types may interfere with results. Hence, there is a need to increase the proportion of neuronal cells in DRG cultures.
Immunopanning is a technique used to purify a variety of cell types, including cortical neurons, astrocytes, oligodendrocyte precursor cells, and microglia. In simple words, it involves adhering an antibody against a cell surface marker to a Petri dish, intended to bind certain cells (Figure 1), and it can be used to select either for or against cell types of interest4,5,6. Immunopanning rat embryonic DRGs to select against non-neuronal cells has been described previously by Zuchero7. However, we have been unable to find an immunopanning protocol specific to mouse DRGs. In this protocol, we build on the basic tenants of the Zuchero protocol, but instead adapted the immunopanning sequence to enrich adult mouse DRG cultures (Figure 2). This is a powerful tool to study established sensory neurons in culture. It is advantageous as it allows for the use of adult mice from genetic, disease, and injury models, so that their sensory neurons may be studied with greater specificity.
This protocol is advantageous for readers looking to increase the proportion of neurons in adult mouse DRG cultures. However, the immunopanning steps can also be omitted, and this protocol can also be used for whole DRG cultures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 um Syringe filters</td>
			<td>Fisher</td>
			<td>723-2520</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dishes</td>
			<td>ThermoFisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well black glass-bottom plates</td>
			<td>Cellvis</td>
			<td>P24-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um cell strainer</td>
			<td>Cedarlane</td>
			<td>15-1070-1(BI)</td>
			<td></td>
		</tr>
		<tr>
			<td>B27+ supplement</td>
			<td>Gibco</td>
			<td>A3582801</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td>for 15% BSA cushion, and ICC (heat shock fraction &#8805;98%)</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A4161</td>
			<td>for 4% BSA for immunopanning, and SATO for media (essentially globulin free, suitable for cell culture, &#8805;99%)</td>
		</tr>
		<tr>
			<td>CD45 antibody</td>
			<td>BD Pharmigen</td>
			<td>550539</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11960069</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Worthington</td>
			<td>LS002007</td>
			<td>for stemxyme solution and panning buffer</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Sigma Aldrich</td>
			<td>D8662</td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Sigma Aldrich</td>
			<td>E6267</td>
			<td>for DNAse solution</td>
		</tr>
		<tr>
			<td>Filter paper P8 grade</td>
			<td>ThermoFisher</td>
			<td>09-795K</td>
			<td>for 8% PFA</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-005-020</td>
			<td>for O4 dish&#160;</td>
		</tr>
		<tr>
			<td>goat-anti-rabbit 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-rabbit IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-005-003</td>
			<td>for PDGFRB dish</td>
		</tr>
		<tr>
			<td>goat-anti-rat IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-005-044</td>
			<td>for CD45 dish</td>
		</tr>
		<tr>
			<td>HBSS -/-</td>
			<td>Sigma Aldrich</td>
			<td>14175145</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td>Sigma Aldrich</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 antibody</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Hybridoma</td>
		</tr>
		<tr>
			<td>Ovomucoid trypsin inhibitor</td>
			<td>Cedarlane</td>
			<td>LS003086</td>
			<td>for low ovo</td>
		</tr>
		<tr>
			<td>paraformaldehyde prills</td>
			<td>Sigma Aldrich</td>
			<td>441244</td>
			<td>for 8% PFA</td>
		</tr>
		<tr>
			<td>PDGFRB antibody</td>
			<td>Abcam</td>
			<td>AB32570</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/ streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma Aldrich</td>
			<td>P8783</td>
			<td>for SATO</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochrloride&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P5780</td>
			<td>for SATO</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Fisher</td>
			<td>S374-1</td>
			<td>for 0.2 M PB</td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>04269</td>
			<td>for 0.2 M PB</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>ThermoFisher</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>Sigma Aldrich</td>
			<td>S5261</td>
			<td>for SATO</td>
		</tr>
		<tr>
			<td>Stemxyme I</td>
			<td>Cedarlane</td>
			<td>LS004107</td>
			<td>for tissue dissociation; combination collagenase</td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma Aldrich</td>
			<td>T1147</td>
			<td>for SATO</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Millipore Sigma</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;3-Tubulin</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td></td>
		</tr>
	</tbody>
</table>

enrichment of adult mouse dorsal root ganglia neuron cultures by immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

The fixed cells stained with DAPI and &#946;3-tubulin were then imaged with a confocal high content screening system. Images were analyzed using suitable commercial software to determine the percentage of DAPI-positive cells that co-labelled with &#946;3-tubulin (Figure 3). Whole DRG cultures were determined to have 42.36% &#177; 6.4% &#946;3-tubulin staining, and immunopanned DRG cultures were determined to have 71.44% &#177;7.43% &#946;3-tubulin staining. This reveals a significant increas...

Watch this Scientific Journal Video about Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning at JoVE.com

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...

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Neuroscience

1.8K Views.  University of Alberta. This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation. 

Video: Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.