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; 表2 ）包被，并将其留在层流罩O / N中。 注意:优选的方法是使用24孔板并仅涂覆中心孔，因为来自外围孔的蒸发趋于更加加速并导致不一致的结果。此外，在t之前，玻璃载玻片可以放置在孔内o涂层。神经元将附着到涂覆的玻璃片上，并可以稍后去除以进行进一步的显微成像。 </li><li>在隔离当天，从井中取出PDL。用无菌水清洗几分钟，然后取出任何剩余的水，并使其干燥1小时（见下文）。 </li><li>干燥后，用层粘连蛋白涂覆载玻片（ 表2 ）。 注意:约10μL层粘连蛋白（10μg/μL）足以涂覆无菌24孔板的每个孔。将其与340μL培养基（总体积:350μL）混合以覆盖整个孔。让它在室温下在培养罩中静置2小时，然后在包被细胞之前吸出。 </li></ol>收获脊髓注意:所有仪器均应进行高压灭菌（135°C和30 psi，4 x 7 min循环），以进行无菌处理。 <ol><li>将1-3天龄的C57BL / 6小鼠小鼠安乐死在带有异氟烷的室内。等待30秒后停止运动，捏腿，确认缺乏反应。 </li><li>使用剪刀将头部与身体分开，小狗处于俯卧位。 </li><li>稳定后腿或手臂在手术台上，背面朝用户。 </li><li>使用弯曲的虹膜剪刀剪掉皮肤。 </li><li>从腰部上方的腰部区切开脊髓，然后切开胸部两侧，将其与身体分开。 注意:这需要从内脏器官仔细解剖脊髓，以避免对其他器官的无意损伤（ 图1a ）。 </li><li>在含有5mL0.2μm过滤器灭菌的磷酸盐缓冲盐水（PBS）的3×10cm培养皿中依次洗涤10s以除去多余的组织。 </li><li>将22 G针头和注射器装满5 mL过滤灭菌的PBS到脊柱的尾端，颅内冲洗，使脐带to退出第四培养皿（ 图1b ）。 </li><li>在15 mL管中收集脊髓，并加入5 mL HABG（ 表1 ）。小心避免压碎脊髓。 </li><li>对垃圾中的每只小狗重复步骤3.1-3.8。 注意:理想情况下，这个过程每个脊髓需要不到30分钟，以确保健康的神经元分离。 </li></ol>隔离神经元注意:以下步骤应在层流罩中进行。预计熟悉基本的无菌技术。 <ol><li> 组织分娩 <ol><li>取包含脊髓的管，轻轻晃动，悬挂组织。 </li><li>将组织从管中倒入60毫米玻璃培养皿和骰子用剃刀刀片，以产生大小为0.5毫米的细碎片。 </li><li>将大孔移液器的组织转移到含有5mL HABG的15mL管中。 </li><li>放在一个30℃水浴30分钟，以使细胞在该温度下平衡。将细胞保持在振动器上，足够使其悬浮在流体中。 注意:此步骤是为了避免在从冰到消化介质转移时对细胞的冲击。保持细胞在30°C有助于减少细胞死亡与另外增加的新陈代谢在37°C。 </li></ol></li><li>准备消化培养基（ 表1 ）。 </li><li> 准备密度梯度（表1）。 <ol><li>在4个不同的15 mL管中制备4层中的每一层，如表1所示 。 </li><li>从每层加入1 mL到一个新的15 mL管中。从底部的第1层开始，并依次添加，直到到达顶层的第4层。加入时避免干扰层。 </li></ol></li><li>从步骤2.2洗涤PDL涂覆的板。用无菌水洗涤数分钟，然后取出任何剩余的水使其干燥1小时。 </li><li> 将组织转移至消化培养基。 <ol><li>从30℃的摇床水浴中取出含有组织的管，并使其沉降数分钟。 </li><li>从37°C水浴中取出消化介质管，然后将其吸入皮肤锁定注射器。 </li><li>从含有组织的管吸出多余的HABG。 </li><li>在注射器上使用一个leur-lock 0.2μm过滤器，将消化液添加到含有组织的管中。 </li><li>将管置于30°C水浴中30分钟。保持细胞晃动刚好足以让它们悬浮在液体中。 注意:重要的是不要将细胞保持在消化介质中太长时间，或者使温度过高，这可能导致过度消化，导致组织悬浮在凝胶状混合物中。 </li></ol></li><li>在此期间，如步骤2.3所示涂覆层粘连蛋白。 </li> &lt;执行研磨（ 即从组织中分离细胞）。 <ol><li>从摇动的30°C水浴中取出管子，使其沉降数分钟。 </li><li>吸出过量消化介质。 </li><li>将组织悬浮于2 mL HABG中。 </li><li>使用狭窄的移液管，捣碎10次，持续45秒。 注意:这可能是最关键的一个步骤，如果不能正确完成，可以显着降低产量。 <ol><li>将组织吸入移液管，并立即将内容物倒空。 </li><li>避免引入空气，因为它会显着降低可行的产量。 注意:理想的移液管是9"玻璃移液管，移液管的顶端应进行火花抛光，使粗糙表面光滑，然后将其放置在二氯二甲基硅烷（DMDCS）在氯仿中的1:20溶液中，然后留下O / N，然后取出移液管，风干用高压釜灭菌。 注意:DMDCS和氯仿是高度易燃的，硅化应在通风橱中进行。 </li></ol></li><li>吸出顶部2 mL上清液，并将其置于标有"收集"的新型15 mL管中。 </li><li>重复步骤4.7.3-4.7.5两次（细胞收集管应至少有6 mL）。 </li><li>将收集管内容物慢慢转移到步骤4.2中制备的梯度管中，避免了梯度的破坏。 </li></ol></li><li>此时，从冰箱中取出以前制备的神经基质培养基（ 表1 ），并使其在37°C浴中温热。 </li><li> 净化神经元。 <ol><li>在800 xg和22℃下离心梯度管15分钟。 </li><li>用移液管收集所需的层（ 图2 ），并置于新的15 mL管中。对于最高纯度的神经n分离（ 即 &gt; 90％），收集层3.为了获得较少的纯度（ 即 &gt; 70-80％），收集层2和3。 </li><li>通过向新收集的层中加入5mL HABG来稀释密度梯度。 </li><li>在22℃下以200xg离心2分钟。 </li><li>弃去上清液，重新悬浮在5 mL HABG中，轻轻摇匀，使细胞悬浮。 </li><li>在22℃离心2分钟200×g。 </li><li>弃去上清液，重悬于3 mL神经巴氏培养基中，轻轻摇匀，重悬细胞。 </li></ol></li><li> 计数细胞 <ol><li>取10μL溶液，现在用神经巴氏培养基中的细胞，并与10μL台盼蓝混合。 </li><li>将10μL该混合物置于玻璃计数室中。 </li><li>使用标准的玻璃计数室，计算四个4×4象限中每一个中的细胞数。添加所有细胞计数（n），多层数2（稀释因子），除以4（计数的象限数），乘以3（神经基底培养基的体积），并乘以10 4以获得细胞/ mL的细胞浓度。 <img alt="方程" src="/files/ftp_upload/55856/55856eq0.jpg" /></li></ol></li><li> 将培养板上的细胞种植 <ol><li>将细胞悬浮液稀释至1毫升神经巴氏培养基中的30万个细胞。 注意:根据上述步骤获得的细胞浓度，加入另外的神经基底培养基以获得3×10 5个细胞/ mL的终浓度。方程式为C 1 V 1 = C 2 V 2 ，其中C 1是从收获获得的细胞的初始浓度; V 1为3mL;如上所述，C 2为3×10 5个细胞/ mL。 V 2求解方程。加入使溶液中细胞总体积相等所需的神经巴培养液体积V 2 。 </li><li>轻轻晃动将细胞分布在溶液中，并向涂覆的24孔板中的每个孔加1 mL。 </li></ol></li></ol>

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作者没有披露。

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><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

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 ...

spinal-cord-neurons-isolation-and-culture-from-neonatal-mice

Research

JoVE Journal

Neuroscience

19.1K 次观看.  University of Colorado Anschutz Medical Campus. 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。 

视频: 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.

基于凝集素的分离和培养的小鼠胚胎运动神经元

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

科研

神经科学

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.

培养小胶质细胞的新生和成年的中枢神经系统

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

免疫与感染

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.

脊髓电

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

生物学

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.

新生儿小鼠脊髓压迫损伤模型

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

医学

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.

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

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.

从胚胎小鼠小脑分离的神经元的主要培养

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.

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 ...

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.

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

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

从成年小鼠脊髓中高效提取星形胶质细胞和小胶质细胞

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.

作者聚焦:从成年小鼠脊髓中分离神经胶质细胞 

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.

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

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

脊髓神经元从新生小鼠中分离和培养

摘要

探索更多视频

基于凝集素的分离和培养的小鼠胚胎运动神经元

培养小胶质细胞的新生和成年的中枢神经系统

脊髓电

新生儿小鼠脊髓压迫损伤模型

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

从胚胎小鼠小脑分离的神经元的主要培养

从产前Isl^mn: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

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于体外测定和转录组学研究

脊髓神经元从新生小鼠中分离和培养

摘要

探索更多视频

基于凝集素的分离和培养的小鼠胚胎运动神经元

培养小胶质细胞的新生和成年的中枢神经系统

脊髓电

新生儿小鼠脊髓压迫损伤模型

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

从胚胎小鼠小脑分离的神经元的主要培养

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

使用微流体腔系统在运动神经元培养物中有机细胞的斧头迁移

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从成年小鼠脊髓中分离纯星形胶质细胞和小胶质细胞，用于 体外 测定和转录组学研究

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