Name: preparation of primary mixed glial cultures from adult mouse spinal cord tissue
Uploaded: 2016-11-19T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue at JoVE.com

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;...

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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?...

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 border="0" cellpadding="0" cellspacing="0" width="2053">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Dulbecco&#8217;s modification of Eagle&#8217;s media (DMEM) with 4.5g/L glucose</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">12-709F</td>
			<td style="width:1168px;">The cDMEM media is a standard, widely used culture media. Individual researcher can decide where to purchase the DMEM and all other component used to make cDMEM media.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">L-Glutamine (100x)</td>
			<td style="width:277px;">Lonza</td>
			<td style="width:119px;">17-605E</td>
			<td>The L-glutamine is a standard, widely used component for various culture media. Individual researcher can decide where to purchase L-glutamine.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Antibiotic-Antimycotic Solution (100x)</td>
			<td style="width:277px;">Corning-Mediatech</td>
			<td style="width:119px;">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 &#181;g/ml amphotericin B. Individual researcher can decide where to purchase individual component.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">2-mercaptoethanol (2-ME)</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td>A BioReagent, suitable for cell culture, molecular biology and electrophoresis.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Papain dissociation system</td>
			<td style="width:277px;">Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>Individual components of this kit can be purchased separately.&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Percoll</td>
			<td>GE Health Care</td>
			<td style="width:119px;">17-0891-01</td>
			<td>Percoll is sold as sterile solution. Undiluted Percoll can be re-autoclaved if needed.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lab-line incubator/shaker&#160;</td>
			<td style="width:277px;">Barstead/Lab-line</td>
			<td style="width:119px;">MaxQ4000&#160;</td>
			<td>This is the incubator/shaker we have currently. Other types of shakers can be used instead.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:489px;">Lipopolysaccharides, Salmonella Minnesota Re595&#160;</td>
			<td style="width:277px;">Sigma-Aldrich</td>
			<td style="width:119px;">L-9764</td>
			<td>Other strains of LPS can also induce glial responses. The magnitude of the responses may vary.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IL-6 DuoSet ELISA&#160;</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY406</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse TNF-alpha DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY410</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse IP-10 (CXCL10) DuoSet ELISA</td>
			<td style="width:277px;">R&#38;D Systems</td>
			<td style="width:119px;">DY466</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse MCP-1 (CCL2) ELISA Opteia set</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">555260</td>
			<td>Each individual Researcher can select the ELISA kit he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Mouse chemokine Q-Plex</td>
			<td style="width:277px;">Quansys Biosciences</td>
			<td style="width:119px;">120251MS</td>
			<td>This product was available for in-house assay at the time. Instead, we sent out samples to the manufacturer for analysis.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">APC-anti-mouse-CD45 (clone 30-F11)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">17-0451</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FITC-anti-mouse-CD11b (clone M1/70)</td>
			<td style="width:277px;">eBioscience</td>
			<td style="width:119px;">11-0112</td>
			<td>Antibody of the same clone but from other vendors can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">Accuri C6 flow cytometer</td>
			<td style="width:277px;">BD Biosciences</td>
			<td style="width:119px;">BD Accuri C6&#160;</td>
			<td>Each individual Researcher can use any flow cytometer he or she prefers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:489px;">FlowJo software</td>
			<td style="width:277px;">Tree Star, Inc.</td>
			<td style="width:119px;">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...

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 ...

The overall goal of this protocol is to establish primary mixed glial cultures from adult mouse spinal cords for in vitro studies. This method provides us an in vitro system to investigate the roles of glial cells in neurological diseases that involve pathological changes within the spinal cord, such as neuropathic pain and multiple sclerosis. The main advantage of this technique is that the glial cultures are prepared from adult mouse spinal cord, providing a system that is more accurately reflecting the in vivo conditions.Demonstrating the procedure will be Jennifer Malon, a technician from my laboratory. Working in a tissue culture hood, transfer four mouse spinal cords into a petri dish of HBSS. Use sterile scissors and forceps to cut each of the spinal cords into small pieces.Then, transfer the pieces to a 50 millimeter conical tube containing Papain DNAse enzyme mixture. Avoid transferring HBSS to the enzyme mixture, as this may result in decreased performance of the enzyme. It is crucial that tissue pieces are well digested to obtain a single cell suspension.However, over digestion will result in fewer viable cells. Each lab needs to perform pilot tests to determine the exact digestion time based on what works best for their cells. Next, gently vortex the tube and then incubate at 37 degrees Celsius for one hour with orbital shaking at 150 rotations per minute.Following the incubation, vortex the tube, and then vigorously triturate the tissue using a five milliliter pipet to promote further dissociation. Then, transfer the cell suspension into a 15 milliliter tube and centrifuge for 300 x g for five minutes at room temperature. During the centrifugation, at 300 microliters of reconstituted albumin ovomucoid inhibitor solution to 2.7 milliliters of ABSS in a sterile tube and mix well.Then add 150 microliters of DNAse solution. Following the centifugation, remove the supernatant, and resuspend the cell pellet in the freshly prepared albumin ovomucoid inhibitor and DNAse solution. Vortex well to break the cell pellet.Then, add three milliliters of reconstituted albumin ovomucoid inhibitor solution, without DNAse, to the cell suspension. Centrifuge the cells at 70 x g for six minutes at room temperature. After the spin, remove the supernatant, which contains membrane fragments, and retain the pellet.To remove myelin from the dissociated spinal cord cells, first add eight milliliters of room temperature 20%gradient density medium into the tube containing the cell pellet and vortex gently. Next, centrifuge the cells at 800 x g for 30 minutes at room temperature without breaking. Following centrifugation, carefully aspirate the top layer of debris, which contains mostly myelin and the supernatant, leaving the pellet.To remove any remaining density gradient, wash the cells by resuspending the pellet with eight milliliters of cDMEM diluted with HBSS. Centrifuge the cells at 400g for 10 minutes at four degrees Celsius. Remove the supernatant, and wash the cells with diluted cDMEM as before.After removing the supernatant, the pellet can be stored on ice until seeding the cells. Once ready for plating, resuspend the cells in 14 milliliters of cDMEM, supplemented with 2-Mercaptoethanol. And add one milliliter of the cell suspension to each well in a 12 well plate.Plate extra wells that can be used to determine the average cell number per well and the microglial content of the culture. Once the cells have been plated, incubate the cells at 35.9 degrees Celsius with 5%carbon dioxide. Change the medium on day 1, which is the day after plating.Some cells are attached to the culture plate, but they are still mostly round. There are also many floating cells and significant debris. Repeat the media change three to four days thereafter until the cells are ready for treatment between days 12 and 14.Mixed glial cells from adult C57 black six mice were cultured at either 37 degrees Celsius or 35.9 degrees Celsius and analyzed via flow cytometry. As you can see, there is no obvious difference in the total cell populations at these temperatures. These representative plots show the CD45 positive CD11b positive microglia populations isolated from the total cell populations previously shown.This figure demonstrates that a higher microglial content can be obtained when mixed glia are cultured at 35.9 degrees Celsius, instead of 37 degrees Celsius. Adult spinal cord, mixed glial cultures were prepared from C67 black six mice. Representative images of the cultured cells at days one, four, eight, and 12 are shown.Images showing the typical progress of the culture, as well as demonstrating the importance of media, change at day one post-culture establishment. Once mastered, the initial set up of mixed glia cell culture can be completed in about four hours, if performed properly. Following the establishment of mixed glia culture, microglia depleted, and microglia enriched cultures, can be obtained from this initial mixed population.However, the yield of microglia enriched cells will be limited unless large number of spinal cords are used to establish cultures. This technique was initially designed to investigate the roles of glial cells during the development of neuropathic pain. However, it can be used to study other neurological diseases that involve pathological changes within the adult spinal cord.

Research

JoVE Journal

Neuroscience

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

斜脊髓切片用于神经前根刺激的制备

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

科研

神经科学

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

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. ...

胚胎小鼠脑混合细胞培养物研究感染和先天免疫

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

作者聚焦：从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫  

从胚胎小鼠大脑中建立混合神经元和神经胶质细胞培养物以研究感染和先天免疫

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...