Name: culturing microglia from the neonatal and adult central nervous system
Uploaded: 2013-08-09T11:00:00
Description: Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

我们要感谢他们的意见和有益的意见的Tsirka实验室成员。这项工作是支持由R01NS42168到SET，12PRE12060489 RB，的NSF-3MT IGERT和特纳论文的奖学金LT，NSF-3MT IGERT JCN。

1。解剖（0天） <ol><li> P0-2鼠标幼仔低温麻醉。与一个Kimwipe浸泡，70％乙醇消毒纸巾擦拭头幼崽。 </li><li>卸下磁头，用一把剪刀，使用4幼仔每10cm的培养板中。在冰冷的Hank氏缓冲液的培养皿中，将磁头。 </li><li>锚头使用弯钳眼窝，并小心地去除皮肤覆盖直microforceps颅骨。 </li><li>取出颅骨与直microforceps，小心使用不穿刺或损坏皮质。最有效的方法是小脑附近，位于头的基础，这个区域将被丢弃起...

<ol>
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E., Tan, S., Wills, T. E., Porritt, M. J., Howells, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+inflammation+in+the+brain+and+spinal+cord+following+mechanical+injury.">Comparison of inflammation in the brain and spinal cord following mechanical injury.</a> J. Neurotrauma. 25, 1217-1225 (2008).</li><li>Schell, J. B., Crane, C. A., Smith, M. F., Roberts, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+ex+vivo+nitric+oxide+production+by+acutely+isolated+neonatal+and+adult+microglia.">Differential ex vivo nitric oxide production by acutely isolated neonatal and adult microglia.</a> J. Neuroimmunol. 189, 75-87 (2007).</li><li>Brannan, C. A., Roberts, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+microglia+from+adult+mice+are+refractory+to+nitric+oxide-inducing+stimuli+due+to+impaired+NOS2+gene+expression.">Resident microglia from adult mice are refractory to nitric oxide-inducing stimuli due to impaired NOS2 gene expression.</a> Glia. 48, 120-131 (2004).</li><li>Tamashiro, T. T., Dalgard, C. L., Byrnes, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+microglia+isolation+from+mixed+glial+cell+cultures+of+neonatal+rat+brain+tissue.">Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.</a> J. Vis. Exp. (66), e3814 (2012).</li><li>Yip, P. K., Kaan, T. K., Fenesan, D., Malcangio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+and+culture+of+primary+microglia+from+adult+mouse+spinal+cord.">Rapid isolation and culture of primary microglia from adult mouse spinal cord.</a> J. Neurosci. Methods. 183, 223-237 (2009).</li><li>Ponomarev, E. D., Novikova, M., Maresz, K., Shriver, L. P., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+culture+system+that+supports+adult+microglial+cell+proliferation+and+maintenance+in+the+resting+state.">Development of a culture system that supports adult microglial cell proliferation and maintenance in the resting state.</a> J. Immunol. Meth. 300, 32-46 (2005).</li><li>Ji, K., Akgul, G., Wollmuth, L. P., Tsirka, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+actively+regulate+the+number+of+functional+synapses.">Microglia actively regulate the number of functional synapses.</a> PLoS One. 8, e56293 (2013).</li><li>Tremblay, M. -. &. #. 2. 0. 0. ;., Majewska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+microglia+in+synaptic+plasticity.">A role for microglia in synaptic plasticity.</a> Comm. Integr. Biol. 4, 220-222 (2011).</li><li>DeWitt, D. A., Perry, G., Cohen, M., Doller, C., Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+microglial+phagocytosis+of+senile+plaque+cores+of+Alzheimer's+disease.">Astrocytes regulate microglial phagocytosis of senile plaque cores of Alzheimer's disease.</a> Expt. Neurol. 149, 329-340 (1998).</li><li>Aloisi, F., Penna, G., Cerase, J., Men&#233;ndez Iglesias, B., Adorini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-12+production+by+central+nervous+system+microglia+is+inhibited+by+astrocytes.">IL-12 production by central nervous system microglia is inhibited by astrocytes.</a> J. Immunol. 159, 1604-1612 (1997).</li><li>Min, K. -. J., Yang, M. -. S., Kim, S. -. U., Jou, I., Joe, E. -. 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Neurosci. 26, 1880-1887 (2006).</li><li>Sasmono, R., Oceandy, D., Pollard, J., Tong, W., Pavli, P., Wainwright, B., Ostrowski, M., Himes, S., Hume, 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+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101, 1155-1163 (2003).</li><li>Ekdahl, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+-+tuning+and+pruning+adult+neurogenesis.">Microglial activation - tuning and pruning adult neurogenesis.</a> Front Pharmacol. 3, 14 (2012).</li><li>Sierra, A., Gottfried-Blackmore, A., McEwen, B., Bulloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile.">Microglia derived from aging mice exhibit an altered inflammatory profile.</a> Glia. 55, 412-424 (2007).</li></ol>

小胶质细胞调节中枢神经系统正常运作，以及各种病症的炎症反应。小胶质细胞突触重塑功能已被牵连在维护正常脑动态平衡15。在神经源性级联，他们参与了海马4，16齿状回神经祖细胞的间隙。因此，有必要制定一个文化系统，在新生儿和成人学习的小胶质细胞，这将覆盖的广袤的发展和成年后，整个小胶质细胞有多个分立功能。我们已经提出了一个快速，高效的小胶质细胞培?...

小胶质细胞是中枢神经系统，最密切相似周巨噬细胞的结构和功能1的的居民免疫细胞的。它最近已表明，出生后的小胶质细胞来源于原始粒细胞前体细胞和胚胎发育的第8天之前产生，颠覆了以前的概念，即出生后的造血祖细胞是小胶质细胞的来源在成人大脑2。他们在一些神经系统疾病中发挥关键作用，并能快速响应感染或受伤的释放促炎或抗炎性细胞因子3。因此，小胶质细胞在中枢神经系统中可以被操纵，影响疾病进展包括一个独立单元。发展强大的和可重复性的方法分离和培养新生儿或成人的小胶质细胞是重要的未来的研究。 小胶质细胞是已知的，在许多的脑疾病是至关重要的玩家。最近，ROL上课新兴的细胞在正常脑小胶质细胞吞噬多余的从1，4海马齿状回神经祖细胞的发育和功能。小胶质细胞也可以调节几个影响脊髓的神经系统疾病，如MS，神经性疼痛，脊髓损伤5-7。脊髓小胶质细胞相比，脑小胶质细胞激活信号，8，9，在本地环境中的差异可能是由于不同的反应。因此，重要的是要建立一个适当的文化研究脊髓小胶质细胞在体外系统。出生的小胶质细胞产生显着的促炎性细胞因子一氧化氮相比，成体细胞在体外刺激后IFN-γ或TNF-α的10,11进一步突出，需要使用成体细胞研究小胶质细胞的某些疾病的情况下。 我们采用的协议，在实验室中立方米lture新生儿小胶质细胞是根据最近的方法，利用摇动混合胶质细胞培养在努力消除小神经胶质细胞的细胞培养瓶12从表面的变化。我们也给出了基于业， 等[13]首次描述了一个协议，从成年小鼠脊髓小胶质细胞培养方法。这种方法提供了更快的方法来培养成年细胞相比其他可用的协议14。将所得的制剂是70％的小胶质细胞，剩余的百分比组成的星形胶质细胞。虽然我们的文化的纯度较其他已发表 ​​的方法13，这种文化系统是有益的探索小胶质细胞在培养应对各种活化刺激，以及为研究疾病，主要影响脊髓强势炎症反应的一个主要特征。 所有描述的协议已获石溪分校学报JOURNALTY IACUC。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>EDTA, 5mM</td>
			<td>Invitrogen</td>
			<td>15567-028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lidocaine, 60mM</td>
			<td>Sigma</td>
			<td>L-5647</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin/EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM, 1X</td>
			<td>Cellgro</td>
			<td>10-017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamycin Sulfate</td>
			<td>Biowittaker</td>
			<td>17-518Z</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 x 10mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly-D-Lysine, 100 &mu;g/ml</td>
			<td></td>
			<td></td>
			<td>Dilute 1:20</td>
		 </tr>
		 <tr>
			<td>HBSS, 1X</td>
			<td>Cellgro</td>
			<td>20-023-CV</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

culturing microglia from the neonatal and adult central nervous system

小胶质细胞是驻地的中枢神经系统（CNS）和巨噬细胞样的细胞，因此，极为重要的作用，在生理和病理过程，如中枢神经系统的成熟发展，多发性硬化，脊髓损伤。小胶质细胞可以激活并招募行动由神经元的损伤或刺激，如性轴索损伤看出，在MS或缺血性脑外伤导致中风。这些免疫功能的中枢神经系统成员也被认为在非病理条件下在突触可塑性的角色。我们采用从新生儿和成人组织的目的是最大限度地存活细胞数，同时最大限度地减少混淆的变量，如其他中枢神经系统的细胞类型和细胞培养碎片的存在下培养的小胶质细胞的协议。我们利用大和容易辨别的中枢神经系统组件（ 如皮质，脊髓节段），这使得整个过程可行，重复性好。成体细胞的使用s是一个合适的替代使用新生儿脑小胶质细胞，，许多病理研究主要影响产后脊髓。这些培养系统也可用于直接测试化合物的效果，可以抑制或促进小胶质细胞活化。由于小胶质细胞活化可以塑造成人中枢神经系统疾病的结果，需要在体外​​系统，在该系统中，新生儿和成人的小胶质细胞可以培养研究。

<html>静息和活化的小胶质细胞的一个例子是图1中所示。小胶质细胞可视化电镀后24小时（ 图1a），表现出分枝形态（休息）。曝光吸试剂，细菌脂多糖（LPS）被激活小胶质细胞形态变化细胞（ 图1b）。 电镀细胞计数的一个例子如图2所示。由于细胞碎片的存在下，DAPI Fluoromount（而不是台盼蓝）溶液中加入等体积的细胞悬浮液?...

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

我们概述方法从新生儿大脑皮层和脊髓成人的可行的小胶质细胞的高效和快速隔离/文化。解剖和电镀皮质小胶质细胞可以在90分钟内完成，与随后发生〜10天的初始剥离后的小胶质细胞的收获。

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 ...

Research

JoVE Journal

Immunology and Infection

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

科研

免疫与感染

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

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 ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

皮层小胶质细胞的分离与保存免疫表型和功能从新生儿鼠

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

在鼻内siRNA输送到中枢神经系统期间放置小鼠的定位装置

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

实验自身免疫性脑脊髓炎中枢神经系统淋巴细胞渗透的流量细胞计量分析

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...