Name: spinal cord neurons isolation and culture from neonatal mice
Uploaded: 2017-07-11T15:00:00
Description: Watch this Scientific Journal Video about Spinal Cord Neurons Isolation and Culture from Neonatal Mice at JoVE.com

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 t...

<ol>
	<li>Qayumi, A. K., 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=Animal+model+for+investigation+of+spinal+cord+injury+caused+by+aortic+cross-clamping.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proximal+arterial+perfusion+pressure+on+function,+spinal+cord+blood+flow,+and+histopathologic+changes+after+increasing+intervals+of+aortic+occlusion+in+the+rat.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocols+for+the+simultaneous+preparation+of+primary+neuronal+cultures+of+the+neocortex,+hippocampus+and+cerebellum+from+individual+newborn+(P0.+5)+C57Bl/6J+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+adult+neurons+and+neurospheres.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hippocampal+neurons+in+dispersed+cell+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+culture+of+spinal+motor+neurons+from+rat+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+motor+neurons+from+the+newborn+mouse+spinal+cord.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocols+for+isolation+of+primary+motor+neurons,+astrocytes+and+microglia+from+embryonic+mouse+spinal+cord.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+preconditioning+protects+against+spinal+cord+ischemia-reperfusion+injury+in+rabbits+by+attenuating+blood+spinal+cord+barrier+disruption.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dependent+protection+of+reseveratrol+against+spinal+cord+ischemic-reperfusion+injury+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+ischemic+preconditioning+protects+the+spinal+cord+against+ischemic+insult:+An+experimental+study+in+a+porcine+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+complications+after+thoracic+aortic+surgery:+long-term+survival+and+functional+status+varies+with+deficit+severity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+spinal+cord+deficits+after+thoracoabdominal+aortic+aneurysm+repair.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-2+agonist+attenuates+ischemic+injury+in+spinal+cord+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+protection+via+alpha-2+agonist-mediated+increase+in+glial+cell-line-derived+neurotrophic+factor.">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>

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 border="0" cellpadding="0" cellspacing="0" width="1357">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Hibernate A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td style="width:107px;">A1247501</td>
			<td style="width:805px;">https://www.thermofisher.com/order/catalog/product/A1247501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hibernate A Minus Calcium - 500 mL</td>
			<td style="width:116px;">Brainbits</td>
			<td>HA-Ca</td>
			<td>http://www.brainbitsllc.com/hibernate-a-minus-calcium/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax 100X - 100 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>35050061</td>
			<td>https://www.thermofisher.com/order/catalog/product/35050079</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement 50X - 10 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>17504044</td>
			<td>https://www.thermofisher.com/order/catalog/product/17504044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Papain, Lyophilized - 100 mg</td>
			<td style="width:116px;">Worthington</td>
			<td>LS003119</td>
			<td>http://www.worthington-biochem.com/pap/cat.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal A Medium - 500 mL</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>10888022</td>
			<td>https://www.thermofisher.com/order/catalog/product/10888022</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>15140122</td>
			<td>https://www.thermofisher.com/order/catalog/product/15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine hydrobromide - 5 mg</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/p6407?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Laminin - 1 mg</td>
			<td style="width:116px;">Thermo-Fisher</td>
			<td>23017015</td>
			<td>https://www.thermofisher.com/order/catalog/product/23017015</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue - 20 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>T8154-20ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/t8154?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OptiPrep Density Gradient Medium - 250 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>D1556-250ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/d1556?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlorodimethylsilane (DMDCS, Sigma Silicoat)</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>440272-100ML</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/440272?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>288306-1L</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glass Pippette - 9&#34;</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:107px;">13-678-20C</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/cls7095d9?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette bulb - 5 mL</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>Z186678-3EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/z186678?lang=en&#38;region=US&#38;cm_sp=Insite-_-prodRecCold_xviews-_-prodRecCold10-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BRAND&#174;&#160;Petri dish, glass - 60x15 mm</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>BR455717-10EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/aldrich/br455717?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile 24 Well Cell Culture Plate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td>M8812-100EA</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/m8812?lang=en&#38;region=US</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hausser Hemacytometer (glass counting chamber)</td>
			<td style="width:116px;">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 height="20">
			<td height="20" style="height:20px;">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 height="21">
			<td height="21" style="height:21px;">NeuN Rabbit Monoclonal Antibody - 100 &#181;L</td>
			<td style="width:116px;">Abcam</td>
			<td>ab177487</td>
			<td>After fixing in paraformaldehyde and blocking with 5% BSA, cells on a 12 mm coverslip were incubated in the antibody diluded to 1:200 for 18 hours in 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Mouse Monoclonal Antibody - 50 &#181;L</td>
			<td style="width:116px;">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 diluded to 1:500 for 18 hours in 4 &#176;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...

Watch this Scientific Journal Video about Spinal Cord Neurons Isolation and Culture from Neonatal Mice at JoVE.com

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 ...

The overall goal of this procedure is to be able to repeatedly and consistently isolate spinal cord neurons that can be used in a variety of experiments. This method can allow us to gain better insight into the biology of spinal cord neurons and hopefully understand their response to a variety of stressors. The main advantage of this technique is, that it allows for the isolation of a significant number of neurons, allowing those neurons to be used in a wide variety of experiments.Visual demonstration of this method is critical as the trituration step where neurons are mechanically isolated from the tissue, requires some time to master, in order to ensure healthy neuron isolation, without killing them. To begin this procedure, separate the head from the body of the euthanized mouse pups, using scissors. Next, stabilize the tail and arms on the procedure table with the dorsal side facing up.Then, cut the skin off using the curved iris scissors. After that, cut the spinal cord from the lumbar region, just above the hips and then cut both sides of the thorax to separate it from the body. Wash the samples sequentially in three 10 centimeter petri dishes, containing five milliliters of sterilized PBS for 10 seconds each, to remove excess tissue.Subsequently, insert a 22 gauge needle and syringe filled with five milliliters of sterilized PBS into the caudal end of the spinal column and flush cranially, allowing the cord to exit into a fourth petri dish. Transfer the spinal cord in a 15 milliliter tube with five milliliters of HABG on ice. Avoid crushing the spinal cord and repeat the procedures for the rest of the pups.Ideally, this process should take no more than 30 minutes for one group of pups, to ensure neuron isolation. To prepare the density gradient, prepare each of the four layers in four 15 milliliter tubes. Then, add one milliliter of each layer into a new 15 milliliter tube.Start with layer one at the bottom, and add sequentially, until reaching layer four at the top. Avoid disturbing the layers while adding and any vigorous movements, that may cause mixing. Next, add the digestion media to the minced tissue.Place the sample in a shaker in the water bath at 30 degrees celsius. Afterward, remove the tissue from the water bath and allow it to settle for a few minutes. To perform trituration, aspirate the excess digestion medium.Suspend the tissue in two milliliters of HABG, using a narrow-bore pipette, triturate 10 times in 45 seconds and avoid introducing air, as it would significantly decrease the viable yield. Trituration is one of the most critical steps of the procedure, the tissue should be carefully drawn into and emptied from the fire polished pipette, avoiding excess vigor which can lead to neuronal death or being too gentle, which would result in a low yield. Following that, aspirate the top two milliliters of the supernatant and transfer it into a new 15 milliliter tube labeled, collection.Repeat the collection procedures two more times, to yield six milliliters of supernatant. Next, slowly transfer the collection tube contents into the gradient tube prepared previously. Avoiding the disruption of the gradient.To purify the neurons, centrifuge the gradient tube for 15 minutes at 800 times G, at 22 degrees celsius. Collect the desired layers and transfer them in a new 15 milliliter tube. For the highest purity neuron isolation, collect layer three.For more yield with less purity, collect layers two to three. Next, dilute the density gradient by adding five milliliters of HABG to the newly collected layers. Centrifuge it, at 200 times G for two minutes at 22 degrees celsius.After two minutes, discard the supernatant and re-suspend the cells in five milliliters of HABG, by flicking the palate. Subsequently, centrifuge the sample at 200 times G for two minutes at 22 degrees celsius again. Then, discard the supernatant and re-suspend the cells in three milliliters of neurobasal medium, by flicking the palate.After that, determine the cell density using a cell counter and then, dilute the suspension to 300, 000 cells per one milliliter of neurobasal medium. Shake gently to distribute the cells and solution and add one milliliter of the sample to each well in the coded 24 well plates. Shown here, is the time course of the neurons in culture, after seeding onto the wells.The cells will typically start adhering to the surface within the first couple of hours. Axons will begin to sprout within the first 24 to 48 hours, connections between various neurons and culture, typically reach maturity at seven days, at which point, experiments are typically carried out on the neurons. In this figure, nuclear staining is shown in blue, with cytoplasmic staining in green and here, the neuron cyctoskeletal protein microtubular associated protein two is stained, showing the outlines and axonal projections of the neurons after a week in culture.The merge images are combined, showing the relative abundance of the neurons. Once mastered, this technique can be done in three to four hours, if it is perform properly. After its development, this technique paves the way for researchers, studying the spinal cord physiology and pathology, to explore the behavior of spinal cord neurons under normal conditions and in response to for pharmacological agents or stressors such as, oxygen and glucose deprivation, just mimicking our surgical procedures.While taking on this procedure, it is important to avoid delaying or prolonging any of the steps to prevent isolation of unhealthy neurons. After watching this video, you should have a good understanding of how to isolate healthy and viable neurons from the spinal cords of neonatal mice that can be cultured in serum free media.

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Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

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

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 ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...