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

作者没有确认。

根据科罗拉多大学机构动物护理和使用委员会的指导方针，对该手术中的动物进行护理和治疗。 准备解决方案<ol><li>在适当的温度下准备并储存所有溶液，如表1所示。 </li></ol> 2.涂层孔和滑块注意：神经元不能很好地粘附在塑料或玻璃表面上。 <ol><li>在分离神经元前一天，用无菌的24孔培养板的孔用0.5mL聚-D-赖氨酸（PDL; <s...

<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>

这种技术允许脊髓神经元的可靠培养。一旦熟练掌握了这项技术，大概需要3.5h才能完成。我们已经能够在大约4小时内从2个独立的小窝（总共16只小鼠）中进行神经元的分离。可行性的关键步骤是能够精通从小鼠中提取脊髓。产量允许电镀几个孔，并能够在各种条件下测试神经元。我们已经能够在成熟后治疗神经元，并使用蛋白质印迹分析可靠地评估蛋白质表达。此外，在孔内使用玻璃载玻片进?...

<html>了解脊髓病理学需要在宏观和微观层面上使用各种模型。大型和小型动物模型1,2,3用于脊髓疾病和损伤的体内研究。 在体内研究这些问题的优点是脊髓分析局限于全脊髓匀浆或组织切片4 。当在其驻留神经元和周围神经胶质中分离脊髓中的特定反应和目标时，这产生了一些歧义。遗传操纵的小鼠越来越多的可用性允许对细胞和分子水平的生物学进行更详细的研究。因此，这里使用新生儿小鼠模型，允许在体外研究脊髓神经元的独特性质和生物学。 在体外分离和维持神经元并不是特别简单，成年啮齿动物的皮层组织的神经元分离技术相对丰富，似乎导致大量的孤立神经元（ 即数百万） 5 ，相反，由于组织质量较小，脊髓组织中神经元的产量较低8,9,10 ，另外在小鼠中，新生儿隔离技术相对较少脊髓神经元和现有的方法受到较低的神经元产量（ 即数百个）的限制9或需要分离胚胎小鼠的费力和资源性重的技术10 。 在这个协议中，我们使用一种技术，允许从新生小鼠脊髓的成本和资源有效的隔离大量的神经元。如以前发表的技术中常见的，我们使用木瓜蛋白酶作为酶蛋白酶，允许从脊髓组织释放神经元5,6 。此外，我们使用密度梯度进行精细细胞分离，其先前已被证明是有效的6,10 。虽然培养细胞的培养基可以根据我们的经验和以前发表的11来改变 ，但补充新鲜的B27培养基补充剂已被证明对神经元寿命至关重要。神经元通常可行达10天，允许进行治疗。

<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

我们提出了脊髓神经元分离和培养的方案。神经元从新生儿C57BL / 6小鼠获得，并在出生后第1-3天分离。一只小鼠垫，通常从一个繁殖对出生的4-10只小狗被聚集一个实验，并且在用异氟烷治疗安乐死后，从每只小鼠单独收集脊髓。将脊柱解剖出来，然后将脊髓从柱上释放出来。然后将脊髓切碎以增加允许神经元和其他细胞从组织中释放的酶蛋白酶的递送的表面积。然后使用研磨将细胞释放到溶液中。随后将该溶液以密度梯度分级，以分离溶液中的各种细胞，允许分离神经元。从一个垃圾组可以分离出约1-2.5×10 6个神经元。然后将神经元接种到涂有粘合剂的孔上rs允许适当的成长和成熟。神经元在生长和培养基中需要约7天达到成熟度，此后可用于治疗和分析。

使用这种技术，单个垃圾（4-10只幼仔）允许分离适合种植在培养板上的1-2.5个10 6个神经元。通常，以上述浓度（ 即 30万个细胞/ mL）接种4-8个孔。 图3显示了在低（ a ）和高（ b ）放大光学显微镜下培养一周后在该浓度下神经元的出现。然而，我们也能够以高达500,000个细胞/孔和低至100,000个细胞/孔的浓度培养?...

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

这项研究提出了一种从WT新生小鼠中分离神经元的技术。它需要从新生小鼠中仔细地解剖脊髓，然后通过机械和酶切割将脊髓组织中的神经元分离出来。

脊髓神经元从新生小鼠中分离和培养

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 ...

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Chicken Embryo Spinal Cord Slice Culture Protocol

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

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

头脊髓和薄片体外制备心律失常活性

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

从产前Islmn：GFP转基因小鼠的奥库罗运动、特罗赫利尔和脊柱运动神经元的分离与培养

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

小鼠完全转型脊髓损伤的诱导

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