The authors have no acknowledgements.

The care and treatment of animals in this procedure were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of Colorado.
1. Preparing Solutions
<ol>
	<li>Prepare and store all solutions at appropriate temperatures, as shown in Table 1.</li>
</ol>
2. Coating Wells and Slides
NOTE: Neurons do not adhere well to plastic or glass surfaces.
<ol>
	<li>One day prior to the isolation of the neurons, coat the wells of a sterile 24-well culture plate with 0.5 mL of Poly-D-Lysine (PDL; Table 2) and leave it in a laminar flow hood O/N. 
	NOTE: The preferred method is to use 24-well plates and to coat only the center wells, as evaporation from the peripheral wells tends to be more accelerated and to lead to inconsistent results. Additionally, glass slides can be placed inside the wells prior to coating. The neurons will attach to the coated glass slides and can be removed later for further microscopic imaging.</li>
	<li>On the day of isolation, remove the PDL from the wells. Wash with sterile water for a few minutes before removing any remaining water and allowing it to dry for 1 h (see below).</li>
	<li>After drying, coat the slides with laminin (Table 2). 
	NOTE: Approximately 10 &#956;L of laminin (10 &#181;g/&#181;L) is enough to coat each well of a sterile 24-well plate. This is mixed with 340 &#956;L of medium (total volume: 350 &#956;L) in order to cover the entire well. Let this sit for 2 h at RT in a culture hood and then aspirate prior to coating the cells.</li>
</ol>
3. Harvesting Spinal Cords
NOTE: All instruments should be autoclaved (135 &#176;C and 30 psi for 4 x 7 min cycles) for sterility.
<ol>
	<li>Euthanize 1-3 day-old C57BL/6 mouse pups in a chamber with isoflurane. Wait 30 s after the cessation of movement and pinch the leg to confirm a lack of response.</li>
	<li>Separate the head from the body using scissors, with pup in the prone position.</li>
	<li>Stabilize the hind legs or tail and arms on the procedure table, with dorsal side facing the user.</li>
	<li>Cut the skin off using curved iris scissors.</li>
	<li>Cut the spinal cord from the lumbar region just above the hips and proceed to cut both sides of the thorax to separate it from the body. 
	NOTE: This requires the careful dissection of the spinal cord from visceral organs to avoid inadvertent damage to other organs (Figure 1a).</li>
	<li>Wash sequentially for 10 s in 3 x 10 cm Petri dishes containing 5 mL of 0.2 &#956;m filter-sterilized Phosphate-Buffered Saline (PBS) to remove excess tissue.</li>
	<li>Insert a 22 G needle and syringe filled with 5 mL of filter-sterilized PBS into the caudal end of the spinal column and flush cranially, allowing the cord to exit into a fourth Petri dish (Figure 1b).</li>
	<li>Collect the spinal cord in a 15 mL tube with 5 mL of HABG (Table 1) on ice. Use care to avoid crushing the spinal cord.</li>
	<li>Repeat steps 3.1-3.8 for each pup in the litter. 
	NOTE: Ideally, this process should take less than 30 min per spinal cord to ensure healthy neuron isolation.</li>
</ol>
4. Isolating Neurons
NOTE: The following step should be performed in a laminar flow hood. Familiarity with basic sterile technique is expected.
<ol>
	<li>Tissue Mincing
	<ol>
		<li>Take the tube containing the spinal cords and shake lightly to suspend the tissue.</li>
		<li>Pour tissue from the tube into a 60 mm glass Petri dish and dice with a razor blade to create fine pieces ~0.5 mm in size.</li>
		<li>Transfer the tissue with a wide-bore pipette into a 15 mL tube containing 5 mL of HABG.</li>
		<li>Place it in a 30 &#176;C water bath for 30 min to allow the cells to equilibrate at this temperature. Keep the cells on a shaker just enough to allow them to be suspended in the fluid. 
		NOTE: This step is done to avoid shocking the cells upon transfer from ice to the digestion medium. Keeping the cells at 30 &#176;C helps to decrease cell death associated with an otherwise increased metabolism at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the digestion medium (Table 1).</li>
	<li>Prepare the density gradient (Table 1).
	<ol>
		<li>Prepare each of the 4 layers in 4 separate 15 mL tubes, as outlined in Table 1.</li>
		<li>Add 1 mL from each layer into a new 15 mL tube. Start with layer 1 at the bottom and sequentially add until reaching layer 4 at the top. Avoid disturbing the layers while adding.</li>
	</ol>
	</li>
	<li>Wash the PDL-coated plates from step 2.2. Wash with sterile water for a few minutes before removing any remaining water and allowing them to dry for 1 h.</li>
	<li>Transfer the tissue to digestion medium.
	<ol>
		<li>Remove the tissue-containing tube from the shaker water bath at 30 &#176;C and allow it to settle for a few minutes.</li>
		<li>Remove the digestion medium tube from the 37 &#176;C water bath and aspirate it into a leur-lock syringe.</li>
		<li>Aspirate off the excess HABG from the tissue-containing tube.</li>
		<li>Use a leur-lock 0.2 &#956;m filter on the syringe to add digestion medium to the tissue-containing tube.</li>
		<li>Place the tube in a 30 &#176;C water bath for 30 min. Keep the cells shaking just enough to allow them to be suspended in the fluid. 
		NOTE: It is important not to keep the cells in the digestion medium for too long or to let the temperature get too high, which could lead to excessive digestion and result in the tissue becoming suspended in a gelatinous mixture.</li>
	</ol>
	</li>
	<li>During this period, coat the laminin as in step 2.3.</li>
	<li>Perform trituration (i.e.&#160;separating the cells from the tissue).
	<ol>
		<li>Remove the tube from the shaking 30 &#176;C water bath and allow it to settle for a few min.</li>
		<li>Aspirate excess digestion medium.</li>
		<li>Suspend the tissue in 2 mL of HABG.</li>
		<li>Using a narrow-bore pipette, triturate 10x for 45 s. 
		NOTE: This is probably the single most crucial step and can significantly decrease the yield if not done properly.
		<ol>
			<li>Aspirate the tissue into the pipette and immediately empty the contents back.</li>
			<li>Avoid introducing air, as it will significantly decrease the viable yield. 
			NOTE: The ideal pipette is a 9&#34; glass pipette. The tip of the pipette should be fire polished to smooth out rough surfaces. It should then be siliconized by placement in a 1:20 solution of dichlorodimethylsilane (DMDCS) in chloroform and left O/N. The pipette should then be removed and allowed to air dry. Subsequently, it should be autoclaved for sterilization. 
			Caution: DMDCS and chloroform are highly flammable, and siliconizing should be carried out in a fume hood.</li>
		</ol>
		</li>
		<li>Aspirate the top 2 mL of supernatant and place it into a new 15 mL tube labeled &#34;collection.&#34;</li>
		<li>Repeat steps 4.7.3-4.7.5 two additional times (the cell collection tube should have 6 mL by the end).</li>
		<li>Slowly transfer the collection tube contents into the gradient tube prepared in step 4.2, avoiding the disruption of the gradient.</li>
	</ol>
	</li>
	<li>At this point, remove the previously prepared neurobasal medium (Table 1) from the refrigerator and allow it to warm in a 37 &#176;C bath.</li>
	<li>Purify the neurons.
	<ol>
		<li>Centrifuge the gradient tube for 15 min at 800 x g and 22 &#176;C.</li>
		<li>Collect the desired layer(s) with a pipette (Figure 2) and place in a new 15 mL tube. For the highest-purity neuron isolation (i.e.&#160;&#62;90%), collect layer 3. For more yield with less purity (i.e.&#160;&#62;70-80%), collect layers 2 &#38; 3.</li>
		<li>Dilute out the density gradient by adding 5 mL of HABG to the newly collected layers.</li>
		<li>Centrifuge at 200 x g for 2 min at 22 &#176;C.</li>
		<li>Discard the supernatant, re-suspend in 5 mL of HABG, and flick the pellet to suspend the cells.</li>
		<li>Centrifuge at for 2 min 200 x g at 22 &#176;C.</li>
		<li>Discard the supernatant, resuspend in 3 mL of neurobasal medium, and flick the pellet to resuspend the cells.</li>
	</ol>
	</li>
	<li>Count the cells.
	<ol>
		<li>Take 10 &#956;L of the solution, now with cells in neurobasal medium, and mix with 10 &#956;L of Trypan blue.</li>
		<li>Place 10 &#956;L of this mixture in a glass counting chamber.</li>
		<li>Using a standard glass counting chamber, count the number of cells in each of the four 4 x 4 quadrants. Add all of the cells counted (n), multiply by 2 (dilution factor), divide by 4 (number of quadrants counted), multiply by 3 (volume of neurobasal medium), and multiply by 104 to obtain the concentration of cells in cells/mL. 
		<img alt="Equation" src="/files/ftp_upload/55856/55856eq0.jpg" /></li>
	</ol>
	</li>
	<li>Seed the cells on culture plates
	<ol>
		<li>Dilute the cell suspension to 300,000 cells in 1 mL of neurobasal medium. 
		NOTE: Based on the concentration of cells obtained in the above steps, additional neurobasal medium is added to obtain a final concentration of 3*105 cells/mL. The equation is C1V1=C2V2, where C1 is the initial concentration of cells obtained from the harvest; V1 is 3 mL; and C2 is 3*105 cells/mL, as discussed above. The equation is solved for V2. Add the volume of neurobasal medium necessary to make the total volume of cells in solution equal to V2.</li>
		<li>Shake gently to distribute the cells in solution and add 1 mL to each well in the coated 24-well plates.</li>
	</ol>
	</li>
</ol>

<ol><li>Qayumi, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+model+for+investigation+of+spinal+cord+injury+caused+by+aortic+cross-clamping%5BTitle%5D)+AND+%22J+Invest+Surg%22%5BJournal%5D)">Animal model for investigation of spinal cord injury caused by aortic cross-clamping.</a> J Invest Surg. 10 (1-2), 47-52 (1997).</li>
<li>Fang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.</a> Int J Mol Sci. 14 (5), 10343-10354 (2013).</li>
<li>Taira, Y., Marsala, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+proximal+arterial+perfusion+pressure+on+function%2C+spinal+cord+blood+flow%2C+and+histopathologic+changes+after+increasing+intervals+of+aortic+occlusion+in+the+rat%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Effect of proximal arterial perfusion pressure on function, spinal cord blood flow, and histopathologic changes after increasing intervals of aortic occlusion in the rat.</a> Stroke. 27 (10), 1850-1858 (1996).</li>
<li>Stoppini, L., Buchs, P. -A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+method+for+organotypic+cultures+of+nervous+tissue%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-182 (1991).</li>
<li>Ahlemeyer, B., Baumgart-Vogt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+protocols+for+the+simultaneous+preparation+of+primary+neuronal+cultures+of+the+neocortex%2C+hippocampus+and+cerebellum+from+individual+newborn+%28P0.+5%29+C57Bl%2F6J+mice%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0. 5) C57Bl/6J mice.</a> J Neurosci Methods. 149 (2), 110-120 (2005).</li>
<li>Brewer, G. J., Torricelli, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+adult+neurons+and+neurospheres%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Isolation and culture of adult neurons and neurospheres.</a> Nat Protoc. 2 (6), 1490-1498 (2007).</li>
<li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+hippocampal+neurons+in+dispersed+cell+culture%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126 (3), 397-425 (1977).</li>
<li>Graber, D. J., Harris, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+culture+of+spinal+motor+neurons+from+rat+embryos%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">Purification and culture of spinal motor neurons from rat embryos.</a> Cold Spring Harb Protoc. 2013 (4), 319-326 (2013).</li>
<li>Anderson, K. N., Potter, A. C., Piccenna, L. G., Quah, A. K., Davies, K. E., Cheema, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+motor+neurons+from+the+newborn+mouse+spinal+cord%5BTitle%5D)+AND+%22Brain+Res+Prot%22%5BJournal%5D)">Isolation and culture of motor neurons from the newborn mouse spinal cord.</a> Brain Res Prot. 12 (3), 132-136 (2004).</li>
<li>Gingras, M., Gagnon, V., Minotti, S., Durham, H. D., Berthod, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+protocols+for+isolation+of+primary+motor+neurons%2C+astrocytes+and+microglia+from+embryonic+mouse+spinal+cord%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Optimized protocols for isolation of primary motor neurons, astrocytes and microglia from embryonic mouse spinal cord.</a> J Neurosci Methods. 163 (1), 111-118 (2007).</li>
<li>Brewer, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal%2C+a+new+serum-free+medium+combination%5BTitle%5D)+AND+%22J+Neurosci+Res%22%5BJournal%5D)">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35 (5), 567-576 (1993).</li>
<li>Fang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption.</a> Int J Mol Sci. 14 (5), 10343-10354 (2013).</li>
<li>Su, M., Zhong, W., Ren, S. <a target="_blank" href="https://www.ajol.info/index.php/tjpr/article/view/139098">Dose-dependent protection of reseveratrol against spinal cord ischemic-reperfusion injury in rats.</a> Trop J Pharm Res. 15 (6), 1225-1233 (2016).</li>
<li>Haapanen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remote+ischemic+preconditioning+protects+the+spinal+cord+against+ischemic+insult%3A+An+experimental+study+in+a+porcine+model%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Remote ischemic preconditioning protects the spinal cord against ischemic insult: An experimental study in a porcine model.</a> J Thorac Cardiovasc Surg. 151 (3), 777-785 (2016).</li>
<li>Conrad, M. F., Ye, J. Y., Chung, T. K., Davison, J. K., Cambria, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+complications+after+thoracic+aortic+surgery%3A+long-term+survival+and+functional+status+varies+with+deficit+severity%5BTitle%5D)+AND+%22J+Vasc+Surg%22%5BJournal%5D)">Spinal cord complications after thoracic aortic surgery: long-term survival and functional status varies with deficit severity.</a> J Vasc Surg. 48 (1), 47-53 (2008).</li>
<li>Wong, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delayed+spinal+cord+deficits+after+thoracoabdominal+aortic+aneurysm+repair%5BTitle%5D)+AND+%22Ann+Thorac+Surg%22%5BJournal%5D)">Delayed spinal cord deficits after thoracoabdominal aortic aneurysm repair.</a> Ann Thorac Surg. 83 (4), 1345-1355 (2007).</li>
<li>Freeman, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alpha-2+agonist+attenuates+ischemic+injury+in+spinal+cord+neurons%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">Alpha-2 agonist attenuates ischemic injury in spinal cord neurons.</a> J Surg Res. 195 (1), 21-28 (2015).</li>
<li>Freeman, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+protection+via+alpha-2+agonist-mediated+increase+in+glial+cell-line-derived+neurotrophic+factor%5BTitle%5D)+AND+%22J+Thorac+Cardiovasc+Surg%22%5BJournal%5D)">Spinal cord protection via alpha-2 agonist-mediated increase in glial cell-line-derived neurotrophic factor.</a> J Thorac Cardiovasc Surg. 149 (2), 578-586 (2015).</li>
</ol>

The authors have no disclosures.

This technique allows for the reliable culture of spinal cord neurons. Once proficiency in the technique is achieved, it takes approximately 3.5 h to complete. We have been able to carry out the isolation of neurons from 2 separate litters (16 mice total) in approximately 4 h. The key step in feasibility is being able to proficiently extract the spinal cords from the mice. The yield allows for plating several wells and for the ability to test the neurons under various conditions. We have been able to treat the neurons af...

Understanding spinal cord pathology requires the use of various models, both on the macroscopic and microscopic levels. Large and small animal models1,2,3 are used for in vivo investigations of spinal cord disease and injury. While studying these issues in vivo has its merits, analysis of the spinal cord is limited to whole spinal cord homogenate or to tissue sections4. This creates some ambiguity when trying to isolate specific responses and targets in the spinal cord among its resident neurons and surrounding glia. The increasing availability of genetically manipulated mice allows for more detailed investigations of the biology at cellular and molecular levels. Thus, a neonatal mouse model is used here, allowing for the study of the unique properties and biology of spinal cord neurons in vitro.
The isolation and maintenance of neurons in vitro is not particularly straightforward. There is a relative abundance of techniques for neuron isolation from the cortical tissue of adult rodents that seem to result in a substantial number of isolated neurons (i.e.&#160;millions)5,6,7. In contrast, the yield of neurons from spinal cord tissue is lower8,9,10, in part due to the smaller mass of tissue. Furthermore, in mice, there is a relative paucity of techniques for the isolation of neonatal spinal cord neurons, and existing methods are limited by lower neuron yields (i.e.&#160;hundreds)9 or laborious and resource-heavy techniques requiring the isolation of embryonic mice10.
In this protocol, we use a technique that allows for the cost- and resource-effective isolation of a substantial number of neurons from the spinal cords of neonatal mice. As is common in previously published techniques, we use papain as an enzymatic protease, allowing for the release of neurons from the spinal cord tissue5,6 . In addition, we use a density gradient for refined cell separation, which has previously been shown to be effective6,10. While the medium in which the cells are incubated can vary, in our experience and as previously published11, supplementation with fresh B27 culture medium supplement has proven to be critical for neuron longevity. The neurons are typically viable for up to 10 days, allowing for treatment to be carried out.

<table><tbody><tr><td>Hibernate A Medium - 500 mL</td><td>Thermo-Fisher</td><td>A1247501</td><td>https://www.thermofisher.com/order/catalog/product/A1247501</td></tr><tr><td>Hibernate A Minus Calcium - 500 mL</td><td>Brainbits</td><td>HA-Ca</td><td>http://www.brainbitsllc.com/hibernate-a-minus-calcium/</td></tr><tr><td>Glutamax 100X - 100 mL</td><td>Thermo-Fisher</td><td>35050061</td><td>https://www.thermofisher.com/order/catalog/product/35050079</td></tr><tr><td>B27 Supplement 50X - 10 mL</td><td>Thermo-Fisher</td><td>17504044</td><td>https://www.thermofisher.com/order/catalog/product/17504044</td></tr><tr><td>Papain, Lyophilized - 100 mg</td><td>Worthington</td><td>LS003119</td><td>http://www.worthington-biochem.com/pap/cat.html</td></tr><tr><td>Neurobasal A Medium - 500 mL</td><td>Thermo-Fisher</td><td>10888022</td><td>https://www.thermofisher.com/order/catalog/product/10888022</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo-Fisher</td><td>15140122</td><td>https://www.thermofisher.com/order/catalog/product/15140122</td></tr><tr><td>Poly-D-Lysine (PDL) hydrobromide - 5 mg</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en&amp;amp;region=US</td></tr><tr><td>Mouse Laminin - 1 mg</td><td>Thermo-Fisher</td><td>23017015</td><td>https://www.thermofisher.com/order/catalog/product/23017015</td></tr><tr><td>Trypan Blue - 20 mL</td><td>Sigma-Aldrich</td><td>T8154-20ML</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en&amp;amp;region=US</td></tr><tr><td>OptiPrep Density Gradient Medium - 250 mL</td><td>Sigma-Aldrich</td><td>D1556-250ML</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en&amp;amp;region=US</td></tr><tr><td>Dichlorodimethylsilane (DMDCS, Sigma Silicoat)</td><td>Sigma-Aldrich</td><td>440272-100ML</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en&amp;amp;region=US</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>288306-1L</td><td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&amp;amp;region=US</td></tr><tr><td>Glass Pippette - 9&quot;</td><td>Sigma-Aldrich</td><td>13-678-20C</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en&amp;amp;region=US</td></tr><tr><td>Pipette bulb - 5 mL</td><td>Sigma-Aldrich</td><td>Z186678-3EA</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en&amp;amp;region=US&amp;amp;cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1</td></tr><tr><td>BRAND&lt;sup&gt;&amp;reg;&lt;/sup&gt;&amp;nbsp;Petri dish, glass - 60 x 15 mm</td><td>Sigma-Aldrich</td><td>BR455717-10EA</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en&amp;amp;region=US</td></tr><tr><td>Sterile 24-Well Cell Culture Plate</td><td>Sigma-Aldrich</td><td>M8812-100EA</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en&amp;amp;region=US</td></tr><tr><td>Hausser Hemacytometer (glass counting chamber)</td><td>Fischer Scientific</td><td>02-671-6</td><td>https://www.fishersci.com/shop/products/hausser-bright-line-phase-hemacytometer-hemacytometer/026716</td></tr><tr><td>Glass Slides - 12 mm sterile cover glass - uncoated</td><td>Neuvitro</td><td>GG-12-1.5-Pre</td><td>http://www.neuvitro.com/german-coverslip-12mm-diameter.htm</td></tr><tr><td>NeuN Rabbit Monoclonal Antibody - 100 &amp;micro;L</td><td>Abcam</td><td>ab177487</td><td>After fixing in paraformaldehyde (PFA) and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluted 1:200 for 18 h in 4 &amp;deg;C</td></tr><tr><td>MAP-2 Mouse Monoclonal Antibody - 50 &amp;micro;L</td><td>Abcam</td><td>ab11267</td><td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluted 1:500 for 18 h in 4 &amp;deg;C</td></tr></tbody></table>

spinal cord neurons isolation and culture from neonatal mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

Using this technique, a single litter (4-10 pups) allows for the isolation of 1-2.5 106 neurons suitable for seeding onto culture plates. Typically, 4-8 wells are seeded at the concentration mentioned above (i.e.&#160;300,000 cells/mL). Figure 3 demonstrates the appearance of neurons at this concentration after a week in culture at low- (a) and high- (b) magnification light microscopy. However, we have als...

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Spinal Cord Neurons Isolation and Culture from Neonatal Mice

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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JoVE Journal

Neuroscience

19.1K Views.  University of Colorado Anschutz Medical Campus.  Deze studie presenteert een techniek voor de isolatie van neuronen van WT-neonatale muizen. Het vereist de zorgvuldige dissectie van het ruggenmerg van de neonatale muis, gevolgd door de scheiding van neuronen uit het ruggenmergweefsel door mechanische en enzymatische splitsing. 

Video: Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Immunology and Infection

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Biology

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Medicine

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 ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies

Source: Eldeiry, M. et al. <a href="https://www.jove.com/video/55856" target="_blank">Spinal Cord Neurons Isolation and Culture from Neonatal Mice.</a> J. Vis. Exp. (2017)
In this video we describe a step-by-step protocol to isolate spinal cord neurons from a neonatal mouse for in vitro studies.

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Figure 1: Spinal column dissected and spinal cord released from a 3 day-old neonatal mouse. The arrow in (a) points towards the caudal end of the spine, where a needle is inserted to release the spinal cord. A released spinal cord (b) is shown submerged in PBS.

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Figure 2: Density gradient, with cells added-on after centrifugation. The layers are outlined and better visualized on the cartoon depiction. The most superficial layer (0) is generally debris from the spinal cord tissue. Layer 1 is rich in oligodendrocytes and astrocytes. Layers 2 and 3 contain the majority of neurons. While layer 3 contains neurons with the highest purity, some supporting cells (i.e., astrocytes and oligodendrocytes) are found in layer 2. The pellet at the bottom mainly contains microglial cells.

Source: Eldeiry, M. et al. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (2017)
In this video we describe a step-by-step ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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