Name: generation of single-cell suspensions from mouse neural tissue
Uploaded: 2009-07-07T19:00:00
Duration: 11 min 17 s
Description: Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

若要在无菌条件下，它是建议执行层流罩中的所有步骤。 1。材料<ul><li>神经组织解离套件（P）（组件：解决方案1,2，3，4，存储缓冲器） </li><li>预分离过滤器</li><li> （可选）髓鞘去除珠</li><li> β-巯基乙醇</li><li> gentleMACS Dissociator </li><li>彗星管</li><li> MACSmix管肩</li><li>的HBSS（W / O） </li><li>的HBSS（W） </li></ul><ol><li>准备开始前的协议以下解决方案： <ol><li>汉克斯“没有二价阳离子的缓冲盐溶液中的Ca 2 +或Mg 2 +（HBSS（W / O ） </li><li> HBSS中，标准的，即与Ca 2 +和Mg 2 +（HBSS（W） </li></ol></li><li>根据不同的抗原表位，在随后的应用程序的兴趣，使用的木瓜蛋白酶为基础的神经组织解离套件（P）或胰蛋白酶为基础的神经组织离解包（T）。 </li><li>从神经组织离解包准备以下解决方案： <ol><li>解决方案2：添加β-巯基乙醇，以0.067毫米</li><li>解决方案4：粉末溶于0.7毫升存储缓冲区（试剂盒提供），轻轻混匀（不要旋涡振荡） </li><li>酶混合物1：移液器1.9毫升溶液中2到C管和孵育10-15分钟50μL解决方案1（套件），在37 ° C。游离于400毫克的脑组织，这是足够的。 </li></ol></li></ol> 2。游离的神经组织<ol><li>称取脑组织中含有1毫升的HBSS（W / O）管</li><li>鼠脑转移到C包含1950年μL预热酶混合1的管。 （注：这个协议是书面为≤400毫克，但卷可以扩展的解决方案，以适应脑组织至1600毫克，C管） </li><li> C管放置到gentleMACS Dissociator和运行方案“m_brain_01”。 </li><li>旋转（4 RPM使用MACSmix管肩）为15分钟，在37 ° C。 </li><li> C管放置到gentleMACS Dissociator和运行方案“m_brain_02”。 </li><li>在解离步骤准备30酶混合，每400毫克组织2液，轻轻混匀20μL的解决方案3和10μL解决方案4。 </li><li>隔密封帽通过添加酶混合物2到C管和无震荡轻轻混匀。 </li><li>孵育37℃10分钟旋转管°孵化器中的C。 </li><li> C管放置到gentleMACS Dissociator和运行方案“m_brain_03”。 </li><li>孵育37℃10分钟旋转管°孵化器中的C。 </li><li>短暂离心收集管底部的样品。 </li></ol> 3。过滤<ol><li>选择一个过滤器大到足以让感兴趣的细胞通过。例如，浦肯野细胞是太大，通过一个30微米的过滤器，但Prominin - 1 +细胞。 </li><li>使用suitable1000μL移液器取出细胞，隔密封帽的形式，通过C管，并将其应用到15毫升收集管上的预分离过滤器。 （注：较大的样本量使用50毫升收集管）。 </li><li>的HBSS（W）10毫升清洗过滤器。 </li><li>离心机在室温下10分钟在300 XG细胞过滤。 </li><li>悬浮在您的介质或为随后的应用程序选择的缓冲区上清。 </li><li>要删除髓鞘碎片，使用髓鞘去除珠。 </li></ol> 4。代表性的结果：请参见图1-3 <img src="/files/ftp_upload/1267/Fig_1.png" alt="图1" /> 图。 1 gentleMACS Dissociator大脑解离，导致97％的活细胞，流式细胞仪分析显示。 <img src="/files/ftp_upload/1267/1267fig2.png" alt="图2" /> 图。 2光镜下神经球形成后7天种植MACS磁性细胞分选后的图片使用反Prominin - 1微珠® NeuroMedium MACS补充B27的PLUS的补充。 CD1小鼠大脑中的神经组织解离套件（P）（P3），准备从细胞。 <img src="/files/ftp_upload/1267/1267fig4.jpg" alt="图3.5" /> 图。 3髓鞘碎片单细胞悬液，大大损害细胞的分离，髓鞘去除珠去除髓鞘碎片，提高了效率，细胞分离。互委会使用反Prominin - 1微球的分离，P22鼠脑分离使用的神经组织解离套件（P）。从单细胞悬液，细胞分离，可以直接使用，或提交髓鞘枯竭使用髓鞘去除珠。比较与无髓鞘去除样品的分离，表明与以前的髓鞘去除样品纯度较高。

<ol>
	<li>Hardt, O., Scholz, C., K&#252;sters, D., Yanagawa, Y., Pennartz, S., Cremer, H., Bosio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+defines+differences+between+region-specific+GABAergic+neurons.">Gene expression analysis defines differences between region-specific GABAergic neurons.</a> Mol Cell Neurosci. 39, 418-428 (2008).</li><li>Lee, J. K., McCoy, M. K., Harms, A. S., Ruhn, K. A., Gold, S. J., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-protein+signaling+10+promotes+dopaminergic+neuron+survival+via+regulation+of+the+microglial+inflammatory+response.">Regulator of G-protein signaling 10 promotes dopaminergic neuron survival via regulation of the microglial inflammatory response.</a> J Neurosci. 28, 8517-8528 (2008).</li><li>K&#246;rnyei, Z., G&#243;cza, E., R&#252;hl, R., Orsolits, B., V&#246;r&#246;s, E., Szab&#243;, B., V&#225;govits, B., Madar&#225;sz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglia-derived+retinoic+acid+is+a+key+factor+in+glia-induced+neurogenesis.">Astroglia-derived retinoic acid is a key factor in glia-induced neurogenesis.</a> FASEB J. 21, 2496-2509 (2007).</li><li>Meylan, F., Davidson, T. S., Kahle, E., Kinder, M., Acharya, K., Jankovic, D., Bundoc, V., Hodges, M., Shevach, E. M., Keane-Myers, A., Wang, E. C., Siegel, 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=The+TNF-family+receptor+DR3+is+essential+for+diverse+T+cell-mediated+inflammatory+diseases.">The TNF-family receptor DR3 is essential for diverse T cell-mediated inflammatory diseases.</a> Immunity. 29, 79-89 (2008).</li><li>Skog, J., W&#252;rdinger, T., van Rijn, S., Meijer, D. H., Gainche, L., Sena-Esteves, M., Curry, W. T., Carter, B. S., Krichevsky, A. M., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+microvesicles+transport+RNA+and+proteins+that+promote+tumour+growth+and+provide+diagnostic+biomarkers.">Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.</a> Nat Cell Biol. 10, 1470-1476 (2008).</li></ol>

作者是德国美天旎生物技术公司，雇员。

gentleMACS Dissociator有利于从一个封闭的系统中的神经组织的单细胞悬浮液的标准化准备。神经组织解离套件进行了优化，保留进一步应用，如immunostainings和免疫细胞的分离1-5需要的抗原表位。在这个协议中，我们展示了鼠标使用gentleMACS Dissociator和神经组织解离套件（P）的大脑的温和酶解，收益率在97％活菌。 5X10 6和1 × 10 7细胞，取决于宿主组织岁之间的神经组织产生100毫克。使用反Prominin - 1微珠针对性Prominin - 1 +细胞进行分离和随后进入的文化。该协议是从小鼠&gt; P7源性神经组织工作时，要额外使用髓鞘去除珠更有效。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C Tubes</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-237</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>gentleMACS&#x2122; Starting Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-235</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>MACSmix&#x2122; Tube Rotator</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-090-753</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx">http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx</a></td>
</tr>
<tr>
<td>Neural Tissue Dissociation Kit (P)</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-628</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx">http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx</a></td>
</tr>
<tr>
<td>Pre-Separation Filters</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-041-407</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx">http://www.miltenyibiotec.com/en/PG_34_247_Pre_Separation_Filters.aspx</a></td>
</tr>
<tr>
<td>Anti-Prominin-1 MicroBeads</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-333</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx">http://www.miltenyibiotec.com/en/PG_654_524_Anti_Prominin_1_MicroBeads.aspx</a></td>
</tr>
<tr>
<td>Anti-Prominin-1-APC</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-092-335</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx">http://www.miltenyibiotec.com/en/PG_599_525_Anti_Prominin_1.aspx</a></td>
</tr>
<tr>
<td>Myelin Removal Beads</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-094-544</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx">http://www.miltenyibiotec.com/en/PG_712_688_Myelin_Removal_Beads.aspx</a></td>
</tr>
<tr>
<td>Art 1000 Reach Pipet Tips </td>
<td>&nbsp;</td>
<td>Molecular BioProducts</td>
<td>Order no. 2079</td>
<td><a href="http://www.mbpinc.com/ASP/products.aspx?query_TipID=61">http://www.mbpinc.com/ASP/products.aspx?query_TipID=61</a> 
<a href="http://www.mbpinc.com/html/products/art/display.html">http://www.mbpinc.com/html/products/art/display.html</a></td>
</tr>
<tr>
<td>Neural Tissue Dissociation Kit (T)</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-231</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx">http://www.miltenyibiotec.com/en/PG_712_656_Neural_Tissue_Dissociation_Kits.aspx</a></td>
</tr>
<tr>
<td>Propidium Iodide Solution</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>Order no. 130-093-233</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx">http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx</a></td>
</tr>		</tbody>
		</table>
		</div>

generation of single-cell suspensions from mouse neural tissue

在神经系统，数百个神经元和神经胶质细胞类型进行了描述。特定的细胞类型中的每个大脑或脊髓有一个细胞表面的分子或分子的决定因素，通过它可以识别和特点的剧目。目前，强大的细胞识别和分离技术的要求，同时限制了细胞的死亡和破坏特征的表面蛋白产生单细胞的准备工作。 gentleMACS Dissociator，胰蛋白酶或木瓜蛋白酶为基础的离解包的组合使用时，可以有效地，轻轻地分解脑组织，同时保留抗原表位，并限制细胞的损失。使用C管和优化，预设的gentleMACS程序实现标准化制备单细胞悬液。一旦产生，单细胞悬浮液是可以治疗的单克隆共轭反Prominin - 1微球，并确定神经祖细胞或纯化进一步使用髓鞘去除珠一样。

Watch this Scientific Journal Video about Generation of Single-Cell Suspensions from Mouse Neural Tissue at JoVE.com

Generation of Single-Cell Suspensions from Mouse Neural Tissue

游离从特定的组织类型细胞需要组织aggitation特定的参数，以获得一个可行的，可培养细胞的高容量。美天旎gentleMACS Dissociator优化这个任务简单，实用的协议。在本出版物中使用本仪器nerual组织进行了解释。

从小鼠神经组织产生单细胞悬浮液

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Research

JoVE Journal

Biology

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

科研

生物学

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

人死后脑组织中的单细胞群获得高品质的RNA

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Kidney Tubuloids from Tissue and Urine

从组织和尿液中产生人类肾脏结核

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

从快速冷冻肝组织中分离细胞核用于单细胞多组学

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

尼罗罗非鱼肠单细胞悬液制备用于单细胞测序

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Isolation and Transcriptome Analysis of Plant Cell Types

植物细胞类型的分离和转录组分析

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...