Name: characterization and isolation of mouse primary microglia by density gradient centrifugation
Uploaded: 2018-02-16T13:30:00
Description: Watch this Scientific Journal Video about Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation at JoVE.com

下列程序已获在莫纳什大学动物伦理学委员会批准。健康未经治疗的新生儿 C57Bl6/J P3-6 小鼠被用来产生代表性的结果。
1. 酶消化
注意: 在分离和培养主细胞时, 考虑不孕是很重要的。虽然确保环境尽可能的不育, 但最初的解剖和小鼠大脑的收获可以在层流流罩之外完成, 随后的所有步骤都在层流罩内进行。
<ol><li>使用无菌器械, 弄死小鼠颈椎脱位, 斩首动?...

<ol>
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小胶质细胞具有支持和抗炎的能力, 通过环境刺激改变。先前的研究表明, 小胶质细胞活化的调节可以赋予神经保护作用。他们为神经元提供保护和修复损伤的能力需要更多的研究来进一步了解这些复杂细胞的现状。因此, 高纯度原发性小胶质细胞的分离是一项重要而有用的技术。这是一个相对较快的方法, 获得高纯度的主要小胶质细胞为体外实验在2天内准备好。
文献?...

围产期期间从炎症、缺氧缺血性和出血中获得的损伤可以有一系列的长期后遗症。围产期脑损伤的复杂病理生理学理论涉及炎症和缺血与随后的神经和轴突死亡1。先天免疫应答在导致伤害的一连串事件中起重要作用2。
小胶质细胞, 中枢神经系统 (CNS) 内的居民免疫单元, 是伤害3的第一个应答者。小胶质细胞是具有保护或毒性能力的塑料电池类型, 依赖于环境4。它们参与趋化、吞噬、抗原表达和细胞因子的产生和活性氧种类的4,5。衰老小胶质细胞不断地对环境进行调查, 并由异物或有害物质的存在4激活。激活会导致一种支持炎症的响应, 在 CNS 保护4中至关重要。这些 M1 的 "亲炎" 表型小胶质细胞主要参与抗原表示和病原体的死亡4。尽管炎症反应在神经保护中起着关键作用, 但不受控制或长时间的炎症可能有害, 导致神经元损伤4。然而, 当暴露在某些环境刺激, 小胶质细胞可以表现出一种抗炎的表型。这些支持修复的 M2 小胶质细胞在伤口愈合和修复过程中具有关键作用6, 释放出一系列 downregulate 炎症、增加吞噬功能和促进修复4, 7. 小胶质细胞的作用多种多样, 包括在髓鞘形成中驱动少突胶质分化 8, 在中风模型的氧和葡萄糖耗尽期间保护神经元9并促进突起在脊髓损伤模型10。
对这些胶质细胞的研究是理解和操作 neuroinflammation 反应的一个重要方面。所述的协议允许进一步研究小胶质细胞调制在 neuroinflammatory 疾病中的治疗潜力。
胶质激活对神经保护角色的调制被观察到了一系列条件11,12,13。因此, 提高当前的理解和进一步研究胶质激活的调制是至关重要的, 需要使用包括体外和体内在内的各种模型。体外研究是一种重要的工具, 因为它们具有更高的效率、更低的成本和调查孤立细胞群体的能力。
文献中描述了一系列的协议, 用于从小鼠脑中分离小胶质细胞, 从而有效地生产出高产样品, 具有良好的生存能力和高纯度。常见的小胶质细胞的分离方法是通过磁分离和长时间晃动的混合胶质细胞培养。通过个人经验发现, 有很大程度的细胞碎片阻碍了磁柱。因此, 使用了下面的协议, 其中包括初始密度梯度离心步骤后, CD11b 磁分离。下面描述的协议已经过优化, 以产生足够数量的高度纯样本。它是有利的由于它的高纯度和短的时间周期-你可以在2天内进行化验, 而无需培养2-3 周。该协议有可能被改编为小鼠星形胶质细胞的分离。

<table border="0" cellpadding="0" cellspacing="0" width="929">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">DMEM, low glucose, pyruvate</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:344px;">11885084</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Antibiotic-Antimycotic (100X)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">DNaseI grade II from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Papain from papaya latex, buffered aqeuous solution</td>
			<td>Sigma-Aldrich</td>
			<td>P3125-100mg</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Percoll</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Hank&#39;s Balanced Salt Solution (1X)</td>
			<td>Gibco</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Hank&#39;s Balanced Salt Solution (10X)</td>
			<td>Gibco</td>
			<td>14185052</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">EasySep Mouse CD11b Positive Selection Kit</td>
			<td>StemCell Technologies</td>
			<td>18770</td>
			<td>EasySep magnet variant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">EasySep magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">EasySep Buffer</td>
			<td>StemCell Technologies</td>
			<td>20144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Dulbecco&#39;s Phosphate buffered saline</td>
			<td>Gibco</td>
			<td>14040182</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Trypsin (2.5%) (10X)</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block&#8482;)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Falcon 5mL Round Bottom High Clarity PP Test Tube, with Snap Cap, Sterile</td>
			<td>Corning</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">175cm&#178; Angled Neck Cell Culture Flask with Vent Cap</td>
			<td>Corning</td>
			<td>431080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Lipopolysaccharides from Escherichia coli O127:B8</td>
			<td>Sigma-Aldrich</td>
			<td>L5024</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">96 Well TC-Treated Microplates size 96 wells, clear, polystyrene, round bottom</td>
			<td>Corning</td>
			<td>CLS3799</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Paraformaldehyde (powder, 95%)</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">Rabbit Anti-Iba1</td>
			<td>Wako</td>
			<td>01919741&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">FACS Antibodies</td>
			<td style="width:133px;">Company</td>
			<td style="width:344px;">Catalog Number</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">V450,Rat,Anti-Mouse,CD45,30-F11,RUO</td>
			<td style="width:133px;">BD Biosciences</td>
			<td>560501</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">PerCP-Cy5.5 CD11b&#160;</td>
			<td>eBiosciences</td>
			<td>45-0112-82</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:285px;">ZombieNIR</td>
			<td>Biolegend</td>
			<td>423105</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">pHrodo Red E. coli BioParticles Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>P35361</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Annexin.V_FITC</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Propodium Iodide solution</td>
			<td>Miltenyi Biotech</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization and isolation of mouse primary microglia by density gradient centrifugation

小胶质细胞, 是大脑中的驻留免疫单元, 是中枢神经系统炎症或损伤的第一反应者。最近的研究发现小胶质细胞是动态的, 能够同时承担炎症和抗炎的表型。两种 M1 (亲炎症) 和 M2 (支持修复) 表型在诸如围产期脑损伤等 neuroinflammatory 条件下起着重要作用, 并表现出不同的功能, 以应对某些环境刺激。胶质激活的调节已被注意到赋予神经保护, 因此提示小胶质细胞可能有治疗潜力的脑损伤。然而, 需要更多的研究, 以更好地了解小胶质细胞在疾病中的作用, 这项议定书有利于这一点。下面描述的协议结合了密度梯度离心过程, 以减少细胞碎片, 与磁性分离, 产生一个高度纯净的主要胶质细胞样本, 可用于体外实验, 没有需要2-3 周的培养。此外, 这些表征步骤产生了关于小胶质细胞的强健的功能数据, 帮助研究, 以更好地了解这两种干细胞的极化和启动, 这在再生医学领域具有很强的意义。

使用此处概述的方法, 可以隔离小胶质细胞的纯种群, 并可以使用体外和资产管制分析来进行表征。首先, 多达18只动物可以使用每一个剔除, 预期产量约 45万-60万胶质细胞。首先确认分离细胞的纯度是至关重要的, 这样做是通过对两个标记 CD45 和 CD11b 的染色来进行的. 胶质细胞的鉴定可以证明是麻烦的, 因为小胶质细胞表达的许多标记也表达了巨噬细胞, 并使用这两个...

Watch this Scientific Journal Video about Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation at JoVE.com

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

本文提出了一种从小鼠脑中分离原发胶质细胞的方法。这种技术有助于促进目前对神经系统状况的理解。将密度梯度离心和磁分离相结合, 产生高纯样品的充分屈服。此外, 我们概述了小胶质细胞的表征步骤。

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

Research

JoVE Journal

Neuroscience

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

科研

神经科学

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Low-Density Primary Hippocampal Neuron Culture

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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

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. ...

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 ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

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 ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

通过磁活化细胞分选在脱髓鞘动物模型中分离小鼠原代小胶质细胞

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...