Name: rapid and refined cd11b magnetic isolation of primary microglia with enhanced purity and versatility
Uploaded: 2017-04-13T12:30:00
Description: Watch this Scientific Journal Video about Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility at JoVE.com

NS088206和ES026892：这项工作是由健康（NIH）全国学院资助。在尤金和琳达·劳埃德主席赋予给AGK和院长教授以AK也被确认。

动物和协议的程序使用批准和机构动物护理和使用委员会（IACUC）在爱荷华州立大学（埃姆斯，IA，USA）的监督 1.混合胶质细胞培养物的种植<ol><li>迅速斩首1-2日龄幼仔用5.5英寸操作剪刀，并立即放置在头冰上的50毫升管中。请注意，这是断头安乐死的方式。 </li><li> 在层流气流罩，使在颅骨和使用4.5英寸直微解剖剪脑膜一个小切口。开始从尾端切割喙端（鼻）。通过使?...

<ol>
	<li>Schulz, C., 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=A+lineage+of+myeloid+cells+independent+of+Myb+and+hematopoietic+stem+cells.">A lineage of myeloid cells independent of Myb and hematopoietic stem cells.</a> Science. 336 (6077), 86-90 (2012).</li><li>Prinz, M., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+brain+macrophages+in+the+molecular+age:+from+origin+to+neuropsychiatric+disease.">Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease.</a> Nat Rev Neurosci. 15 (5), 300-312 (2014).</li><li>Alliot, F., Godin, I., Pessac, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derive+from+progenitors,+originating+from+the+yolk+sac,+and+which+proliferate+in+the+brain.">Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain.</a> Brain Res Dev Brain Res. 117 (2), 145-152 (1999).</li><li>Ransohoff, R. M., Cardona, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myeloid+cells+of+the+central+nervous+system+parenchyma.">The myeloid cells of the central nervous system parenchyma.</a> Nature. 468 (7321), 253-262 (2010).</li><li>Kettenmann, H., Kirchhoff, F., Verkhratsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+new+roles+for+the+synaptic+stripper.">Microglia: new roles for the synaptic stripper.</a> Neuron. 77 (1), 10-18 (2013).</li><li>Su, X., 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=Synuclein+activates+microglia+in+a+model+of+Parkinson's+disease.">Synuclein activates microglia in a model of Parkinson's disease.</a> Neurobiol Aging. 29 (11), 1690-1701 (2008).</li><li>Dzamko, N., Geczy, C. L., Halliday, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+is+genetically+implicated+in+Parkinson's+disease.">Inflammation is genetically implicated in Parkinson's disease.</a> Neuroscience. 302, 89-102 (2015).</li><li>Xing, B., Bachstetter, A. D., Van Eldik, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+p38alpha+MAPK+is+critical+for+LPS-induced+neuron+degeneration,+through+a+mechanism+involving+TNFalpha.">Microglial p38alpha MAPK is critical for LPS-induced neuron degeneration, through a mechanism involving TNFalpha.</a> Mol Neurodegener. 6, 84 (2011).</li><li>Panicker, N., 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=Fyn+Kinase+Regulates+Microglial+Neuroinflammatory+Responses+in+Cell+Culture+and+Animal+Models+of+Parkinson's+Disease.">Fyn Kinase Regulates Microglial Neuroinflammatory Responses in Cell Culture and Animal Models of Parkinson's Disease.</a> J Neurosci. 35 (27), 10058-10077 (2015).</li><li>Moehle, M. S., West, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M1+and+M2+immune+activation+in+Parkinson's+Disease:+Foe+and+ally?.">M1 and M2 immune activation in Parkinson's Disease: Foe and ally?.</a> Neuroscience. 302, 59-73 (2015).</li><li>Saijo, K., Glass, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+origin+and+phenotypes+in+health+and+disease.">Microglial cell origin and phenotypes in health and disease.</a> Nat Rev Immunol. 11 (11), 775-787 (2011).</li><li>Paolicelli, R. C., 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=Synaptic+pruning+by+microglia+is+necessary+for+normal+brain+development.">Synaptic pruning by microglia is necessary for normal brain development.</a> Science. 333 (6048), 1456-1458 (2011).</li><li>Floden, A. M., Combs, C. 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+repetitively+isolated+from+in+vitro+mixed+glial+cultures+retain+their+initial+phenotype.">Microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype.</a> J Neurosci Methods. 164 (2), 218-224 (2007).</li><li>Saura, J., Tusell, J. M., Serratosa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+isolation+of+murine+microglia+by+mild+trypsinization.">High-yield isolation of murine microglia by mild trypsinization.</a> Glia. 44 (3), 183-189 (2003).</li><li>Gordon, 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=A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia.">A simple magnetic separation method for high-yield isolation of pure primary microglia.</a> J Neurosci Methods. 194 (2), 287-296 (2011).</li><li>Prinz, M., Priller, J., Sisodia, S. S., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+CNS+myeloid+cells+and+their+roles+in+neurodegeneration.">Heterogeneity of CNS myeloid cells and their roles in neurodegeneration.</a> Nat Neurosci. 14 (10), 1227-1235 (2011).</li><li>Marek, R., Caruso, M., Rostami, A., Grinspan, J. B., Das Sarma, ., J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+cell+sorting:+a+fast+and+effective+method+of+concurrent+isolation+of+high+purity+viable+astrocytes+and+microglia+from+neonatal+mouse+brain+tissue.">Magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue.</a> J Neurosci Methods. 175 (1), 108-118 (2008).</li><li>Hasko, G., Pacher, P., Vizi, E. S., Illes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+receptor+signaling+in+the+brain+immune+system.">Adenosine receptor signaling in the brain immune system.</a> Trends Pharmacol Sci. 26 (10), 511-516 (2005).</li><li>Radulovic, M., Yoon, H., Wu, J., Mustafa, K., Scarisbrick, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+thrombin+receptor+modulates+inflammation+and+astrogliosis+to+improve+recovery+after+spinal+cord+injury.">Targeting the thrombin receptor modulates inflammation and astrogliosis to improve recovery after spinal cord injury.</a> Neurobiol Dis. 93, 226-242 (2016).</li><li>Ay, M., 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=Molecular+cloning,+epigenetic+regulation,+and+functional+characterization+of+Prkd1+gene+promoter+in+dopaminergic+cell+culture+models+of+Parkinson's+disease.">Molecular cloning, epigenetic regulation, and functional characterization of Prkd1 gene promoter in dopaminergic cell culture models of Parkinson's disease.</a> J Neurochem. 135 (2), 402-415 (2015).</li><li>Gordon, 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=Protein+kinase+Cdelta+upregulation+in+microglia+drives+neuroinflammatory+responses+and+dopaminergic+neurodegeneration+in+experimental+models+of+Parkinson's+disease.">Protein kinase Cdelta upregulation in microglia drives neuroinflammatory responses and dopaminergic neurodegeneration in experimental models of Parkinson's disease.</a> Neurobiol Dis. 93, 96-114 (2016).</li></ol>

旧版小胶质细胞的分离方法具有有限的回收率关于各种蛋白通过Western印迹分析和RNA通过qRT-PCR分析是不恰当的。差分的粘附性和温和的胰蛋白酶消化的方法是具有低的小神经胶质收率13，14，15两种常用的方法。基于列的CD11b的方法也有低回收率，但实现了比差的粘附性和温和的胰蛋白酶消化<sup...

小胶质细胞是中胚层起源的MYB-亚独立定居巨噬细胞，其选自c-kit + / CD45 -分化erythromyeloid在卵黄囊1，2的血岛祖细胞。一旦胚胎的小胶质细胞已定植的中枢神经系统（CNS），它们从一个变形虫过渡到网状形式3。因为它们的动态后果探测潜在的侮辱4良性脑实质这些成人小胶质细胞被列为监视者。虽然小胶质细胞只向CNS细胞群的约10％，其以瓦能力彼此之间确保了实质4,5的最大扫描。危险相关的分子模式（DAMP的），如α突触核蛋白6,7和淀粉样蛋白β8，或对athogen相关分子模式（PAMP），如脂多糖（LPS）9，经典激活的小胶质细胞，以促进其特征在于，回复到变形虫活动状态，产生一氧化氮，肿瘤坏死因子α（TNFα），白介素1β的炎症反应（IL-1β），IL-6，IL-12，和趋化因子CC基序配体2 9，10，11。在神经炎性病症，例如帕金森氏病，其中致病α突触核蛋白积累了，神经变性周期从多巴胺能神经元，其中释放出更多的聚集α突触核蛋白的死亡创建的，进一步促进小胶质细胞7的经典活化。类似于周巨噬细胞，小神经胶质细胞还可具有在抗炎细胞因子IL-4和IL-10的存在下交替地激活的能力，给他们的有效的促进神经修复和衰减炎症2，11 IAL。除了在中枢神经系统的免疫作用，小胶质细胞已被开发过程中修剪突触描述为神经回路的重要调节器。例如，Cx3cr1- KO小鼠具有较少致密的小胶质细胞和减少突触修剪，这导致树突棘，未成熟的突触，并不发达CNS 12的电图案的过剩。在CNS中的稳态了解这些生理复杂性和小胶质细胞的各种功能性作用是对寻求治疗剂靶向神经变性疾病的关键。 在神经免疫学领域， 在体外实验是因为机理研究更大的可行性非常可取的，较低的维护成本，并为是少时间和劳动密集。 Furthermo再，以分离细胞群的能力是在规定条件下以描绘那些靶细胞的功能性是至关重要的。无数小胶质细胞的分离方法存在，但它们可通过它们，以获得相对高的数量和纯度为广泛的实验13，14，15的能力的限制。例如，分化11b的簇（细胞CD11b）是单核细胞，巨噬细胞，小胶质细胞和16的共同的表面标志物。通过利用细胞CD11b，磁分离的方法最初被描述为每新生儿脑17产生了基于列的办法〜99.5％的纯度，和〜1.6×10 6的小胶质细胞。我们实验室最近开发了一种无柱的CD11b磁分离方法15，我们在聚苯乙烯管通过用缀合藻红蛋白（PE）的单克隆抗体标记的CD11b进行。一种双特异性第二ARY抗体到PE和与PE葡聚糖复合物。一旦结合，葡聚糖包被的磁性颗粒被引入，其结合所述抗体复合物的葡聚糖端。最后，聚苯乙烯管放置在小神经胶质细胞隔离的磁铁。这种方法增加了一倍的产率每〜新生儿脑，但在减少纯度〜97％的成本3.2×10 6的小胶质细胞。 在本文中，我们展示了一个快速和精制无柱的CD11b磁选协议（ 图1）。由于CD11b的磁分离试剂盒的价格是一样的这种改进的方法仍然是我们原来的无柱的方法是可行的。完成时间减半，这可以最大限度地提高细胞的存活和产量至关重要的减少。值得注意的是，从这个优化的方法实现纯度〜&gt; 99％，比从我们的实验室15开发的原始无柱方法实现的纯度显着改善。最重要的是，细胞CD11b-PE没有被利用，从而无需从光孵育远，并允许荧光显微镜使用红色通道的。最后，如在原来的CD11b的方法，用这种改进的方法获得高产率和高纯度的星形细胞级分。星形胶质细胞是中枢神经系统中最大量的胶质细胞，导致的想法，他们的自我平衡的功能有关的病理生理学18不可缺少的。这些胶质细胞在不同的生理功能，如形成血 - 脑屏障，提供营养支持，维持神经递质的动态平衡，响应损伤神经保护作用形成胶质疤痕，学习和记忆，和神经炎症，体现他们的调查潜在的胶质中发挥作用生物学19。形态与小胶质细胞和星形胶质细胞的功能都通过激光共聚焦显微镜，免疫印迹，实时定量聚合酶链反应（定量RT-PCR），G被确定里斯亚硝酸盐测定中，和Luminex多重细胞因子测定。由这个协议所提供的精细化提供更高的置信度涉及小胶质星形细胞或纯度，与红色信道的可用性荧光显微镜的更广泛的应用，并节省了时间，所有这些都是在体外实验重要。

<table>
	<tbody>
		<tr>
			<td>EasySep CD11b Separation Kit II</td>
			<td>StemCell Technologies</td>
			<td>18970</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1x)</td>
			<td>Life Technologies&#160;</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Life Technologies&#160;</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids (100x)</td>
			<td>Life Technologies&#160;</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (100x)</td>
			<td>Life Technologies&#160;</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>13H469</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco by Life Technologies</td>
			<td>25200</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>Gibco by Life Technologies</td>
			<td>14190250</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and refined cd11b magnetic isolation of primary microglia with enhanced purity and versatility

小胶质细胞是主要的反应，以中枢神经系统损伤;然而，还有许多关于他们在调节神经炎症的作用尚不清楚。小胶质细胞是功能类似于在巨噬细胞炎性测量应力中胚层细胞。经典的（M1型）和替代（M2型）的巨噬细胞的激活也已经扩展到小神经胶质细胞中是为了更好地理解下面的相互作用这些表型具有在神经炎性病症​​，例如帕金森氏，阿尔茨海默氏症，和亨廷顿氏病。 体外实验利用初级小胶质细胞提供了可以扩展到体内环境的快速和可靠的结果。虽然这是在体内实验具有明显的优势，隔离的小胶质细胞而实现最佳的纯净，足够的产量一直是个挑战。目前使用的常用方法无论是从回收率低，纯度低，或两者受苦。在此，我们高程模型onstrate该实现中的时间量的一半高的细胞回收和提高的纯度的无柱的CD11b磁分离方法的改进。我们建议这种方法优化为主的小胶质细胞分离为研究神经炎症和神经退行性病变的目的非常有用的模型。

小神经胶质细胞使用细胞CD11b阳性选择试剂盒分离II具有高纯度原代小鼠的小胶质细胞使用上述方案分离并接种于聚-D-赖氨酸包被的盖玻片上，以检查隔离的纯度。每孔和免疫细胞化学分析铺板万个细胞使用离子化的钙结合适配器分子1（Iba1）作为小胶质细胞和神经胶质纤维酸性蛋白（GFAP）作为星形胶质细胞的标记物的标记物以?...

Watch this Scientific Journal Video about Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility at JoVE.com

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

这里，我们提出用于体外实验的协议来隔离来自出生后小鼠幼仔的小胶质细胞（1天）。此简易分离方法产生了高收率和纯度，在替代方法一个优点显著允许阐明小胶质生物学的目的范围广泛的实验。

原代小胶质的快速和精细的CD11b电磁隔离带增强的纯度和多功能性

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

Research

JoVE Journal

Neuroscience

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

磁共振波谱的用途，用于对初级运动皮层新陈代谢双半球经颅电刺激效果的测量工具一

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

科研

神经科学

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

鼠小胶质细胞的分离培养促进无血清培养基动态分支形态学的研究

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

来自成人大鼠尿膀胱的原发神经元和胶质的隔离与培养

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

从新生小鼠中磁分离小胶质细胞用于原代细胞培养

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

大鼠星形胶质细胞和小胶质细胞的原代培养及其在肌萎缩侧索硬化症研究中的应用

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...