Adult spinal cord mixed cultures were first developed using rats with grant support from an NIH/NIDA R01 award (PI De Leo) and an NIH T32 training grant (PI Green). These methods were further adapted for mouse spinal cords with support from the following grants: NIH/NIDA 5K01DA023503 (PI Cao), NIH/NINDS 5R21NS066130 (PI Cao), and NIH/NIGMS P20GM103643 (PI Meng). The authors would also like to thank Dr. Alejandro M. S. Mayer, Department of Pharmacology, Chicago College of Osteopathic Medicine, for his technical help in obtaining microglia-rich mixed glial cells.

下面的协议被批准的机构动物护理和使用委员会（IACUC）在新英格兰的大学。以下协议是从4成年小鼠脊髓制备胶质细胞培养。 1.在无菌条件下文化胡德溶液的制备<ol><li>制备培养基:Eagle氏培养基（cDMEM）含DMEM的完全Dulbecco氏修改（用4.5克/升葡萄糖），10％胎牛血清（FBS），2mM的L-谷氨酰胺，100IU / mL青霉素，100毫克/毫升链霉素，250纳克/毫升两性霉素B，和50μM2-巯基乙醇（2-ME;见1.1.1）。混合和过滤消毒所有其他组件，然后添加FBS。存储培养基在4℃ <ol><li>制备50mM的2-ME /磷酸盐缓冲盐水（PBS）原液:添加100μL的浓缩2-ME（14.3 M）到28.6毫升1×PBS中，并过滤消毒（该溶液可以储存在4℃下数月）。 üSE 1毫升的2-ME原液每毫升1000培养基。 </li></ol></li><li> 密度梯度介质的制备 <ol><li>准备一个股票等渗密度梯度媒体解决方案（ 例如，100％珀）从1份10X常规无菌PBS和9份无菌股票密度梯度介质（ 如，珀）。 </li><li>稀释定期1×无菌PBS的100％的密度梯度介质（在1.2.1。制造），使20％的密度梯度介质（ 例如，10毫升100％的密度梯度介质的用40mL 1×PBS中），并储存在4 ℃。制备细胞培养物之前，使20％的密度梯度介质至室温（RT）。 </li></ol></li><li> 解决方案从木瓜蛋白酶解离系统准备 注: 木瓜蛋白酶解离系统所用的材料表来识别。其它相似的解离试剂盒（包括在内部装配）也可以使用。所有COMPONENTS必须是无菌的。 <ol><li>添加32毫升的Earle氏平衡盐溶液（EBSS）中的卵类粘蛋白的抑制剂混合物（粉末）。 </li><li>加入5毫升的EBSS的一个木瓜蛋白酶小瓶（粉末）。将在37℃水浴中的木瓜蛋白酶小瓶10分钟或直至木瓜蛋白酶溶解。 </li><li>添加EBSS 500微升一个DNA酶小瓶（粉）。轻轻混匀。 </li><li>添加脱氧核糖核酸酶溶液（以上）的木瓜蛋白酶小瓶的250微升。 </li><li>计算所需的上述木瓜蛋白酶/ DNA酶的混合物（每鼠脊髓木瓜蛋白酶/ DNA酶混合物的约800微升）的合计量和所需要的混合物转移到50 mL管中用于组织消化（步骤3.2）。存储在原始小瓶剩在4℃至少两周。 </li><li>保持冰从包的所有组件，直到需要。 </li></ol></li><li>制备脊髓收集管:一种无菌的15毫升管用Hank氏平衡盐溶液（HBSS 5mL的;从所述木瓜蛋白酶解离系统，0;收集脊髓）和一个无菌的15毫升管1X PBS（对于填补了5毫升注射器，见步骤2.4）。此外，5毫升的HBSS准备一份无菌培养皿英寸（35 mm或60毫米），保持它在培养罩。 </li></ol> 2.脊髓收藏注:所有设备必须是无菌的。由IACUC批准的指定区域执行收集。 <ol><li>获得收获小鼠脊髓以下工具:直锐/钝器18厘米的手术剪，一个12厘米的标准手术刀（＃3固体），碳钢无菌手术刀片＃10，小动物斩首，12厘米的标准曲线钳，和无菌20G针连接到一个5毫升的注射器。 </li><li>安乐死用CO 2对动物和消毒鼠标头并用70％乙醇（EtOH中）的后区。 </li><li>动物斩首的刽子手。就后腰中间纵切口，显露肌肉层。使通过在臀部的水平脊椎动物列干净的横断与直无菌手术剪（18厘米）（通过覆盖脊椎动物列肌肉层也切）。 </li><li>将动物对新鲜一次性擦拭片。小心地将20G的针（连接到一个5毫升的注射器填充有无菌1×PBS中（步骤1.4））到脊柱朝喙侧。按住向下动物紧密，迅速推向注射器。整个脊髓应该从子宫颈年底问世。收集脊髓到含有HBSS 15毫升管。 </li><li>重复步骤2.4小鼠的其余部分。每只动物之间，消毒用70％乙醇所有使用的仪器。 注意:通常情况下，一个单一的12孔板可以为每4小鼠脊髓（见4.3）成立。 </li></ol> 3.单细胞悬液的制备注:执行步骤3和4的培养罩把一切都无菌。 <ol><li>传输所有脊髓入含有HBSS的皮氏培养皿（步骤1.4）。削减每个脊髓成许多精美的，小件用无菌剪刀和镊子。转移脊髓组织用无菌镊子或10毫升吸管含有制备的木瓜蛋白酶/ DNA酶的酶混合物（步骤1.3.5）的50-mL锥形管（避免加入HBSS的酶混合物，因为这将进一步稀释制备酶溶液，并可能导致酶的性能降低）。 </li><li>然后振荡试管混合。在37℃下1小时管在培养箱/摇床圆周振荡在150转。 </li><li>涡管再次大力磨碎用5毫升吸管进一步推动分离组织中的酶溶液。 </li><li>在RT细胞悬液转移到15毫升管中并离心，在300×g离心5分钟。 </li><li>在离心过程中，混合2.7毫升EBSS与重组白蛋白卵类粘蛋白INH的300微升在无菌管ibitor溶液（1.3.1）。加入DNA酶溶液（步1.3.3）的150微升。 </li><li>离心后，除去上清，悬浮与上述制备（步骤3.5）的溶液中的细胞沉淀。涡流以及打破细胞沉淀。 </li><li>添加3mL的重组白蛋白的卵类粘蛋白抑制剂溶液（步骤1.4.1），以细胞悬浮液。离心细胞，在70×g离心在RT 6分钟。除去上清液（包含膜碎片）。 </li></ol>从单细胞悬液髓鞘4.进一步去除<ol><li>在800 XG在RT轻轻加入8毫升20％的密度梯度介质（在步骤1.2.2制备）到含有细胞沉淀管中，涡流破坏沉淀，离心所述细胞30分钟无制动 。小心除去碎片（主要是髓鞘）和上清液的顶层，但保留沉淀。 </li><li>要除去密度梯度的残余，由resuspendi洗细胞纳克用8毫升稀释cDMEM的（1份cDMEM和2份HBSS）中的细胞沉淀。离心在400×g离心10分钟，将细胞在4℃除去上清液并以相同的方式与稀释cDMEM（上文）再次洗涤细胞。保持冰的细胞，直到他们播种。 </li><li>除去上清，重悬在培养基中的细胞沉淀（cDMEM供给与2-ME（在步骤1.1中制备））。对于一个12孔板中，使用3毫升×4（使用小鼠数）+ 2毫升= 14毫升介质。这将确保有对整个板足够的细胞悬浮液（12个孔），并提供了可用于确定每孔的平均细胞数和培养物的小胶质含量额外井。如果将被用于其它类型的培养容器，按比例计算所需的培养基的总体积。 </li><li>添加1毫升细胞悬液到12孔平板的每个孔中。 </li><li>孵育细胞在含有5％CO 2 35.9 摄氏度 。 </li><l我&gt;第D 1更改媒体（通过抽吸去除旧的媒体），然后每3 - 4天之后（通常是改变D中的媒体:1，4，8，11，然后用细胞第12天）。 注:在第1天，将培养可以含有由于从脊髓组织中的残余物髓鞘显著的碎片;因此，建议改变第1天传媒（请参阅有关更多信息的讨论）。培养物准备好D之治疗12 - 14细胞通常是在D 12 80％汇合，并可以用D 14通常为接近100％汇合时，对D 12，有每孔约100,000细胞在12孔平板。 </li></ol><img alt="图4" src="/files/ftp_upload/54801/54801fig4.jpg" /> 图2:在不同时间建立的混合胶质细胞培养物混合后的神经胶质细胞的代表性图像从成年C57BL / 6小鼠中制备初级脊髓混合胶质。代表图像显示神经胶质培养的电镀后的进展。在第1天，一些细胞附着到培养板上，但它们仍然是多为圆形。也有许多漂浮细胞和显著碎片。在第4天，大部分细胞附着到培养板上。细胞似乎网状可见的过程。细胞零星分布和文化在这个时候约20-30％汇合。在第8天，文化是50-60％之间的融合。文化的部分地区有细胞的大片。文化的其他领域有细胞的生长稀疏。补丁中的某些细胞采取了"广场样"外观。第12天，培养物是在等于或高于80％汇合（此处示出的图像中的约90％汇合）。细胞在此时生长的对数期，天之间12-14是使用胶质细胞进行实验的最佳时间。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54801/54801fig4large.jpg" target="_blank">请点击这里查看</a>更大的版本这个数字。 <ol start="7"><li>检查使用通过标准荧光激活细胞分选（FACS）染色方案11从额外孔细胞（步骤4.3），使用抗小鼠CD45抗体和抗小鼠的CD11b单克隆抗体的组合中的小胶质细胞的内容。 CD45 +细胞CD11b +细胞被识别为小胶质细胞。 </li></ol>

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The authors have nothing to disclose.

它是执行所有步骤，包括媒体，以获得实验之间重复性的结果改变时间表-以一致的方式非常关键。下面的步骤是获得优良的品质成人混合胶质尤为关键。 酶消化是该协议的一个重要方面。至关重要的是，该组织片是为了获得单细胞悬浮液充分消化。然而，过度消化将导致显著较少的细胞。每个实验需要执行导频测试，以确定基于所述类型用于孵化小瓶的确切消化时间，每次使用的平均组织量，并摇动器的类型（旋转与轨道摇床）。此外，无论所选择的木瓜蛋白酶解离系统的，有必要持续使用相同的组件由相同的源，作为组件的偶然替换可能导致产量显著变化的细胞的总数。 在这个协议中，髓鞘是使用密度梯度介质中除去。它是使和自这些溶液的密度是温度敏感在RT使用密度梯度溶液的关键。密度梯度溶液通常保存在4℃。如果需要的话，该密度梯度溶液可在室温下设置的培养的前一天保持过夜。此外，以下在20％的密度梯度的细胞的30分钟离心，将细胞应立即从离心机中取出，以防止大的碎片从移（ 即，移动从溶液的顶部离开;参见步骤4.1）。 这对文化的D 1后开始清除杂物是很重要的。虽然细胞将存活，并且如果培养基被改变每3到4天，改变第D 1个媒体将提供更多的"静态"和"清洁"培养以后增殖。这个允许细胞在不太干扰的环境下成长，并且细胞可以保持在培养的第21天后开始。 对于每个实验的小神经胶质内容可在此之前使用混合胶质进行审查。即使在最一致的技术，小胶质含量可能仍然显著实验之间变化。有些细胞效应依赖于小胶质含量4,10;因此，关键是监视每个组建立培养物的小胶质含量。从这种做法获得的数据可以帮助解释实验之间的变化，以及提供新型关于星形细胞与小胶质细胞的特定实验条件下的贡献（有时意外）知识。此外，在小胶质细胞含量的季节性变化已观察到。这可能是策划大型成套实验时，需要考虑的另外一个因素。此外，虽然通常的神经元会不是我们的培养条件下生存，作为的NeuN（一个neuronal-标记）阳性细胞没有被在我们的胶质细胞培养免疫组织化学染色后观察到的，培养可以含有少突胶质细胞的数量有限的（这还没有被常规量化）。之一应该决定该人群是否需要根据具体的实验条件进行审查。 与许多实验方法，这种方法可以根据个人研究人员的需要进行修改。重要的是要执行导频实验全面测试之前，常规地使用它们的具体的修改。此外，应该指出的是，混合的神经胶质可用于获得小胶质贫和小胶质细胞富集的培养物研究星形细胞显性与特定刺激后的小胶质细胞显性细胞的反应，如前对新生儿混合胶质细胞3所述， 4，但是，小胶质细胞富集的细胞的产率将除非大量的脊限定线用于在建立之初的文化。这是这种方法的一个限制，尤其是如果使用的小鼠。当使用大鼠脊髓，一个个体脊髓（代替4-小鼠脊髓）可用于设置一个12孔板中。最后，小胶质含量不出现是小鼠和大鼠成人脊髓神经胶质培养物之间显著不同4,5两者小鼠和大鼠成人脊髓胶质细胞培养产生的细胞因子产生方面到LPS刺激一个强有力的反应。然而，当使用替代刺激可以观察到不同的反应。 总之，这种啮齿类动物成年胶质培养系统提供了另一种方法来研究神经胶质细胞在体外 。从该培养系统更紧密地获得的结果反映了将在体内条件下可观察与从新生儿培养物或细胞系获得的结果。在目前的方法中，神经胶质细胞是C从成年啮齿类ollected相比，新生儿神经胶质4（ 图1）时成年胶质响应于非常不同刺激。细胞没有通过永生化的程序，这将被用于产生和维持的细胞系显著操纵。虽然这种方法最初被开发在神经性疼痛的研究中使用，它可以被用来研究涉及脊髓内的病理变化，如ALS和MS其他神经系统疾病。

慢性疼痛是一种严重的健康问题，影响到在美国的约100万成年人，具有高达635 $ 1十亿预计每年成本。越来越多的证据表明脊髓神经胶质细胞的神经性疼痛，最具破坏性的种慢性疼痛2中的一个发展的显著贡献。一个适当的体外培养体系将在细胞和分子水平深入研究神经胶质细胞的作用显著帮助。 目前，研究人员使用的体外神经胶质培养系统包括从新生小鼠或大鼠大脑皮质组织来源主要是神经胶质细胞和永生化从新生小鼠衍生的神经胶质细胞系或大鼠原胶质细胞。新生细胞含有未分化细胞显著数字，可以进一步分化成神经胶质细胞（主要为星形胶质细胞和小胶质细胞），从而提供一bundant细胞实验使用3。然而，我们以前的研究已经表明，新生儿脑胶质细胞从成人脊髓神经胶质细胞在脂多糖（LPS）刺激的反应显著不同。例如，白细胞介素（IL）-4显示的增强的LPS诱导的一氧化氮的抑制作用（NO）产生的成年大鼠脊髓神经胶质相比新生儿大脑胶质4。此外，趋化因子产生刺激后的由神经肽的型材，降钙素基因相关肽（CGRP），是新生小鼠脑部的神经胶质细胞之间无可争议不同（在参考文献中描述的方法。5）和成年小鼠脊髓神经胶质细胞（ 图1）。建立的细胞系是易于使用和维护，可以在很短的时间内提供大量的细胞。在一般情况下，永生化细胞系中产生或者使用病毒介导的永生化系统（如广泛使用的小胶质细胞系BV2）6,7或以下第自发变换电子识别（如星形胶质细胞系的C8-D1A和小胶质细胞系的C8-B4）8,9的细胞系具有优异的学习星形细胞和小胶质细胞单独的分子特征。然而，从细胞系所获得的结果总是需要在原代细胞或在体内条件进一步验证。还应当指出的是，没有发生过一个从啮齿动物的脊髓神经胶质衍生的神经胶质细胞系的报告。 以帮助调查脊髓神经胶质细胞中的神经性疼痛，是从成年小鼠脊髓衍生已通过适应用来产生成年大鼠混合胶质细胞培养4先前报道的方法开发了一种培养系统，5的发展中的作用该小鼠脊髓神经胶质文化进一步细化最近10和更详细的这篇文章中介绍。成人脊髓混合胶质细胞培养Ç一个与来自适合的特定的研究的需要选出的菌株的小鼠建立的，并且细胞能够在培养物中维持长达21天后的培养开始。这种方法可以在神经性疼痛的研究中使用，以及在涉及脊髓内的病理变化，如肌萎缩性侧索硬化症（ALS），人免疫缺陷病毒（HIV）相关的感觉神经病，和多个各种神经疾病的调查硬化症（MS）。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modification of Eagle&amp;rsquo;s media (DMEM) with 4.5 g/L glucose</td><td>Lonza</td><td>12-709F</td><td>The cDMEM media is a standard, widely used culture media. Individual researchers can decide where to purchase the DMEM and all other components used to make cDMEM media.</td></tr><tr><td>L-Glutamine (100x)</td><td>Lonza</td><td>17-605E</td><td>The L-glutamine is a standard, widely used component for various culture media. Individual researchers can decide where to purchase L-glutamine.</td></tr><tr><td>Antibiotic-Antimycotic Solution (100x)</td><td>Corning-Mediatech</td><td>30-004-CI</td><td>This is a combination of penicillin, streptomycin and Amphotericin formulated to contain 10,000 units/mL penicillin G, 10 mg/mL streptomycin sulfate and 25 &amp;micro;g/mL amphotericin B. Individual researchers can decide where to purchase individual components.</td></tr><tr><td>2-mercaptoethanol (2-ME)</td><td>Sigma-Aldrich</td><td>M3148</td><td>A BioReagent, suitable for cell culture, molecular biology and electrophoresis.</td></tr><tr><td>Papain dissociation system</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td>Individual components of this kit can be purchased separately.&amp;nbsp;</td></tr><tr><td>Percoll</td><td>GE Health Care</td><td>17-0891-01</td><td>Percoll is sold as sterile solution. Undiluted Percoll can be re-autoclaved if needed.</td></tr><tr><td>Lab-line incubator/shaker&amp;nbsp;</td><td>Barstead/Lab-line</td><td>MaxQ4000&amp;nbsp;</td><td>This is the incubator/shaker we have currently. Other types of shakers can be used instead.</td></tr><tr><td>Lipopolysaccharides, &lt;em&gt;Salmonella Minnesota&lt;/em&gt; Re595</td><td>Sigma-Aldrich</td><td>L-9764</td><td>Other strains of LPS can also induce glial responses. The magnitude of the responses though, may vary.</td></tr><tr><td>Mouse IL-6 DuoSet ELISA&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>DY406</td><td>Each individual Researcher can select the ELISA kit he or she prefers.</td></tr><tr><td>Mouse TNF-alpha DuoSet ELISA</td><td>R&amp;amp;D Systems</td><td>DY410</td><td>Each individual Researcher can select the ELISA kit he or she prefers.</td></tr><tr><td>Mouse IP-10 (CXCL10) DuoSet ELISA</td><td>R&amp;amp;D Systems</td><td>DY466</td><td>Each individual Researcher can select the ELISA kit he or she prefers.</td></tr><tr><td>Mouse MCP-1 (CCL2) ELISA Opteia set</td><td>BD Biosciences</td><td>555260</td><td>Each individual Researcher can select the ELISA kit he or she prefers.</td></tr><tr><td>Mouse chemokine Q-Plex</td><td>Quansys Biosciences</td><td>120251MS</td><td>This product was not available for in-house assay at the time. Instead, we sent out samples to the manufacturer for analysis.</td></tr><tr><td>APC-anti-mouse-CD45 (clone 30-F11)</td><td>eBioscience</td><td>17-0451</td><td>Antibody of the same clone but from other vendors can be used.</td></tr><tr><td>FITC-anti-mouse-CD11b (clone M1/70)</td><td>eBioscience</td><td>11-0112</td><td>Antibody of the same clone but from other vendors can be used.</td></tr><tr><td>Accuri C6 flow cytometer</td><td>BD Biosciences</td><td>BD Accuri C6&amp;nbsp;</td><td>Each individual Researcher can use any flow cytometer he or she prefers.</td></tr><tr><td>FlowJo software</td><td>Tree Star, Inc.</td><td>FlowJo7.6.5</td><td>Each individual Researcher can use any analysis software he or she prefers.</td></tr></tbody></table>

preparation of primary mixed glial cultures from adult mouse spinal cord tissue

它已被广为接受的脊髓神经胶质反应显著到神经性疼痛的发展作出贡献。已通过体外细胞培养系统中获得关于在细胞和分子水平胶质活动巨大的信息。所用的体外系统，主要包括从新生儿脑皮质组织来源的原神经胶质细胞和永生化细胞系。然而，这些系统可能无法反映体内脊髓神经胶质细胞的特性。为了使用培养体系，更好地反映体内状况进一步调查脊髓神经胶质细胞在周围神经损伤引起的神经性疼痛发展中的作用，我们的实验室已经发展到建立原发性脊髓混合胶质细胞培养从方法成年小鼠。简单地说，脊髓从成年小鼠收集，并通过木瓜蛋白酶消化后髓鞘去除与加工窝点性梯度网上平台。单细胞悬浮液是在完整的Dulbecco补充有2-巯基乙醇（2-ME）改良的Eagle培养基（cDMEM）培养在35.9℃。这些培养条件为混合胶质细胞的生长进行了优化。在这些条件下，细胞准备用于12之间实验 - 14天（细胞通常是在这段时间对数期）建立培养物（D 0）之后，并且可以被保持在培养条件达到D 21。该培养系统可以用于研究脊髓神经胶质细胞的刺激时与各种物质和试剂的反应。除了神经性疼痛，这种系统可用于研究在涉及脊髓神经胶质细胞的病理改变其他疾病胶质反应。

该方法可用于从小鼠和大鼠制备混合胶质细胞。每孔中的培养的D 12启动后一个12孔板的平均总细胞数应该是相对稳定的，与每孔周围100,000个细胞时细胞从小鼠脊髓的。从该方法获得的神经胶质细胞可以在被设计为检查时的物质和感兴趣剂施用成人脊髓神经胶质反应实验中使用。 图3提供了从一个典型的实验中的细胞因子和趋化因子反应的例子，其中成人脊髓小胶质细胞从成年BALB / c小鼠获得，并与在培养的d- 13启动后的LPS（ 沙门氏菌明尼苏达 Re595）处理。收集上清液用商业上availab后24小时LPS处理用于经由酶联免疫吸附测定（ELISA）的细胞因子和趋化因子水平的测定乐包。 当第一次成立这样的成人神经胶质培养体系，细胞在一个标准的文化环境， 在 37℃下保温10％ - 在该状态下，一般的小胶质含量从5不等。然而，有人指出，尽管使用了一致的培养技术，文化会在某些情况下，要么非常低的小胶质含量（&lt;2％）或相对较高的小胶质含量（15 - 20％）10。它可以是令人沮丧的获得有极少数小胶质细胞时的实验结果依靠混合培养中的小胶质细胞反应的文化。继亚历杭德罗·梅耶MS博士（药理学教研室，骨科医学学院芝加哥）12的建议下，该方法被越来越多的混合神经胶质细胞在35.9 摄氏度修改这导致更一致的和更高的小胶质细胞产量（范围10之间 - ％40和余下大部分芳ound 20％）的大多数培养物的范围内。这一改进在图4中示出如先前报道，小胶质估计，以弥补5 -中枢神经系统的神经胶质人口的21％成年小鼠13-15。虽然这两个培养条件提供了包含小胶质细胞的相似量的作为体内估计培养物中，改性培养条件（35.9℃）是更合适的实验中，它是检查由两个星形胶质细胞和小胶质细胞的反应是至关重要的。 <img alt="图1" src="/files/ftp_upload/54801/54801fig1.jpg" /> 图 1: 降钙素基因相关肽（CGRP）诱导的趋化因子生产通过混合胶质细胞从新生儿BALB / c小鼠的大脑（左）或成年BALB / c小鼠的脊髓制备混合胶质细胞（右）与各种剂量的治疗CGRP的。在培养物上清液的几个趋化因子的水平<html>通过在最佳时机多重检测（由制造商执行）确定（平均值±SEM，N = 2 - 6）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54801/54801fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54801/54801fig2.jpg" /> 图3:混合神经胶质在小鼠成人脊髓细胞因子和趋化因子应答的LPS刺激从BALB / c小鼠中制备并用各种剂量的LPS刺激成人脊髓混合胶质细胞。 IL-6的（A），肿瘤坏死因子（TNF）-α（B）中，干扰素γ诱导蛋白10（IP-10，也称为CXCL10）（℃），和单核细胞趋化蛋白1的水平（MCP- 1，也被称为CCL2）（D）在培养物上清液在24小时后LPS处理通过ELISA来测定。ecsource.jove.com/files/ftp_upload/54801/54801fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54801/54801fig3.jpg" /> 图 4: 在成人混合胶质细胞培养小胶质含量从成年C57B6 / L的小鼠产生混合的神经胶质细胞，并在任一37℃或35.9℃。培养从每个培养小胶质含量通过流式细胞仪分析用的APC的抗小鼠CD45抗体（克隆30-F11）和FITC-抗小鼠的CD11b（克隆M1 / 70）。从流式细胞分析代表性的图被示出。从培养的总细胞群中小区A（37℃）和C（35.9℃），和小胶质细胞群体进行鉴定（CD45 +细胞CD11b +细胞）从B中的各自的总细胞群进一步分离（37℃）和D（35.9℃）。进行流式细胞仪分析，如前所述5进行分析的所有数据。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54801/54801fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue at JoVE.com

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

神经性疼痛的发展涉及脊髓神经胶质细胞的病理变化。从成年脊髓组织的，设计用于在体外研究这些细胞中的胶质可靠的培养体系缺乏。因此，我们在这里展示了如何建立从成年小鼠的脊髓组织的主要胶质细胞混合培养。

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 ...

preparation-primary-mixed-glial-cultures-from-adult-mouse-spinal-cord

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.5K 次观看.  University of New England. 神经性疼痛的发展涉及脊髓神经胶质细胞的病理变化。从成年脊髓组织的，设计用于在体外研究这些细胞中的胶质可靠的培养体系缺乏。因此，我们在这里展示了如何建立从成年小鼠的脊髓组织的主要胶质细胞混合培养。 

视频: Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

科研

免疫与感染

Derivation of Glial Restricted Precursors from E13 mice

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

从E13小鼠胶质限制前体的推导

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors ...

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 ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

共培养 AdMSC 和神经胶质细胞以减少朊病毒诱导的炎症

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments.
One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation.
A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

作者聚焦:深入了解朊病毒病研究进展

Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth ...

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 ...

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

摘要

探索更多视频

此视频中的章节

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

从E13小鼠胶质限制前体的推导

培养小胶质细胞的新生和成年的中枢神经系统

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

Primary Microglia Isolation from Postnatal Mouse Brains

脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症

脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症

脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症

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

第一次混合胶质细胞培养的成年小鼠脊髓组织准备

摘要

探索更多视频

此视频中的章节

初级小胶质细胞的分离，从新生大鼠脑组织胶质细胞混合文化

从E13小鼠胶质限制前体的推导

培养小胶质细胞的新生和成年的中枢神经系统

从多个中枢神经系统区域解剖和分离小鼠神经胶质细胞

Primary Microglia Isolation from Postnatal Mouse Brains

脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症

脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症

脂肪来源的间充质基质细胞与原代混合胶质细胞共培养，以减少朊病毒诱导的炎症

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

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