Name: enrichment of adult mouse dorsal root ganglia neuron cultures by immunopanning
Uploaded: 2023-02-24T14:15:00
Description: Watch this Scientific Journal Video about Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning at JoVE.com

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 concent...

<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>

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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