Name: enrichment of adult mouse dorsal root ganglia neuron cultures by immunopanning
Uploaded: 2023-02-24T14:15:00.000+00:00
Duration: 8 min 57 s
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 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>

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	<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><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 &amp;ge;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, &amp;ge;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&amp;nbsp;</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&amp;nbsp;</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>&amp;beta;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...

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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 ...

enrichment-adult-mouse-dorsal-root-ganglia-neuron-cultures

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JoVE Journal

Neuroscience

1.9K Views.  University of Alberta. Dit protocol stelt ons in staat om het aantal neuronen in volwassen muis dorsale wortel ganglia culturen te verbeteren. Dit kan helpen bij het bepalen van de bijdrage van neuronale cellen aan een bepaalde respons. We hebben een immunopanningstap toegevoegd aan een basis DRG-kweekprotocol.Het belangrijkste voordeel is om te selecteren tegen niet-neuronale cellen. Als je nieuw bent in primaire DRG-culturen, is het het beste om dissecties te oefenen om zoveel mogelijk DRG's te krijgen en ...

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

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

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 ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA

Source: Dong, X., et.al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57547">Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis.</a>&#160;J. Vis. Exp.&#160;(2018)
In this video, we demonstrate the chromatin immunoprecipitation technique to identify protein binding sites on specific regions of the genomic DNA of oligodendrocyte precursor cells via the selective immunoprecipitation of protein-bound chromatin fragments. This method helps to study the interaction of several regulatory proteins, including transcription factors involved in gene regulation.

Source: Dong, X., et.al., Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

Neuronal Enrichment of Dorsal Root Ganglia by Immunopanning

Source: Maguire, A. D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64603">Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning.</a> J. Vis. Exp. (2023)
This video demonstrates a protocol for enriching neuronal cultures from dorsal root ganglia by immunopanning, thereby improving their purity and utility for neurological research.

1. Experimental method

	Day 1: preparation of dishes
	
		In a biosafety cabinet, coat the desired culture surface with enough poly-D-lysine to cover the ...

Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-culturing of a Dorsal Root Ganglion Explant with Schwann Cells for Neuron Myelination

Source: Blusch, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64768">In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.</a> J. Vis. Exp. (2023)
This video demonstrates the coculturing model of a dorsal root ganglion explant and Schwann cells. In coculture conditions, ascorbic acid promotes the differentiation of Schwann cells into myelinating forms. Myelinating Schwann cells wrap the axons multiple times to create a myelin sheath, which is essential for nerve function.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Schwann cell culture

	...

Isolation of DRG Neurons and Coculture with Schwann Cell Precursors to Generate Schwann Cells

To harvest the dorsal root ganglia, transfer the first embryo into a 10-centimeter culture dish containing room-temperature PBS under a dissecting microscope in the prone position and insert micro-dissecting forceps along either side of the spinal cord.
Using blunt dissection, separate the spinal cord from the surrounding soft tissue, using forceps to excise the spinal cord along the neck opening and tail stub. Remove the residual soft tissue from the freed spinal cord until only the spinal cord, nerve roots, and attached dorsal root ganglion remain.
Detach the individual dorsal root ganglia from their connecting nerve roots. Then, use a pipette pen equipped with a 1-milliliter pipette tip to transfer up to 100 dorsal root ganglia into a 1.5-milliliter microcentrifuge tube of PBS.
Collect the ganglia by centrifugation, and re-suspend the pellets in 200 microliters of recombinant enzymatic cell dissociation reagent per tube. After 10 minutes at 37 degrees Celsius, centrifuge the dorsal root ganglia again using a 200-microliter pipette tip to gently triturate the pellets in dorsal root postganglionic neuron maintenance medium.
Seed the dorsal root ganglion cells at 5 x 103 cells per square centimeter onto Poly-D-Lysine laminin-coated six-well plates in 1.5 milliliters of dorsal root ganglion neuron maintenance medium per well.
After two days in culture at 37 degrees Celsius and 5% CO2, wash the cells and return the neuronal cell cultures to the incubator in fresh dorsal root ganglion neuron purification medium for another two days.
After 3 to 4 maintenance and purification cycles, the dorsal root ganglion cell cultures should test positive for the neuronal marker Tuj-1 and negative for the glial cell marker S100 beta.
On day 7 of the Schwann cell-like cell culture, rinse the cells in PBS, followed by dissociation with 0.5 milliliters of recombinant enzymatic cell dissociation reagent per well at 37 degrees Celsius for 5 minutes. Resuspend the cell pellet in co-culture medium and seed the Schwann cell-like cells onto the purified dorsal root ganglion neuron culture at 1 x 103 cells per square centimeter for 14 days of co-culture at 37 degrees Celsius and 5% CO2.

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

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