Name: neuronal nuclei isolation from human postmortem brain tissue
Uploaded: 2008-10-01T00:00:00
Description: Watch this Scientific Journal Video about Neuronal Nuclei Isolation from Human Postmortem Brain Tissue at JoVE.com

作者感谢理查德博士Konz和工作人员在美国马萨诸塞大学医学院的流式细胞仪核心设施提供咨询和技术支持。也可用于糖尿病内分泌研究中心授予DK032520支持的核心资源。这项工作是由美国国立精神卫生研究所（NIMH），国家药物滥用研究所（NIDA）和国家儿童健康和人类发展研究所的赠款支持。

细胞核提取<ol><li>于5 mL裂解液1 250毫克冷冻死后脑组织douncer置于冰上。要收到约500万的排序后流式细胞仪细胞核，我们通常使用1000毫克每样本的组织。 </li><li>一旦该组织已经解冻，dounce为1分钟，而冰的组织。 </li><li>匀浆组织，放入15毫升明确超速离心机管。管置于冰上。 </li><li>以9 mL蔗糖溶液2中，用移液器移液器明确超速离心机管底部，并释放 2蔗糖溶液与蔗糖溶?...

<ol>
	<li>Siegmund, K. D., Connor, C. M., Campan, M., Long, T. I., Weisenberger, D. J., Biniszkiewicz, D., Jaenisch, R., Laird, P. W., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+the+human+cerebral+cortex+is+dynamically+regulated+throughout+the+life+span+and+involves+differentiated+neurons.">DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.</a> PLoS ONE. 2, 895-895 (2007).</li><li>Jiang, Y., Matevossian, A., Huang, H. S., Straubhaar, J., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., Frisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Acevedo, L. G., Iniguez, A. L., Holster, H. L., Zhang, X., Green, R., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+ChIP-chip+analysis+using+10,000+human+cells.">Genome-scale ChIP-chip analysis using 10,000 human cells.</a> Biotechniques. 43, 791-797 (2007).</li></ol>

这里介绍的协议应重点研究神经细胞染色质的表观遗传变化，更普遍，特别是有用的，对神经细胞的细胞核，在正常的发展和老龄化，或在神经或精神疾病，分子表型。当脑组织细胞的异质性，包括潜在的变化在老化过程中神经元的神经胶质细胞的口粮，或因疾病是关注1，该方法是特别的优势。例如，通过染色质免疫沉淀技术结合目前的排序协议，我们最近在大脑衍生神经营养因子（BDNF）基因启动子的...

试剂： <table><tr><th colspan="3" align="left"> 1。裂解液</th></tr><tr><td> 0.32M </td><td>蔗糖</td><td> 5.47摹</td></tr><tr><td> 5毫米</td><td> 氯化钙 </td><td> 250μL </td></tr><tr><td> 3毫米</td><td> mg（醋酸酯）2 </td><td> 150μL </td></tr><tr><td> 0.1毫米</td><td> EDTA </td><td> 10μL </td></tr><tr><td> 10MM </td><td>的Tris - HCl，PH8 </td><td> 500μL </td></tr><tr><td> 1毫米</td><td>数码地面电视</td><td> 17μL </td></tr><tr><td> 0.1％ </td><td> TRITON X - 100 </td><td> 50μL </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0">调整蒸压水容至50 ml </td></tr></table><table><tr><th colspan="3" align="left"> 2。蔗糖溶液</th></tr><tr><td> 1.8米</td><td>蔗糖</td><td> 30.78摹</td></tr><tr><td> 3毫米</td><td> mg（醋酸酯）2 </td><td> 150μL </td></tr><tr><td> 1毫米</td><td>数码地面电视</td><td> 17μL </td></tr><tr><td> 10毫米</td><td>的Tris - HCl，PH8 </td><td> 500μL </td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0">调整蒸压水容至50 ml </td></tr></table><table><tr><th colspan="3" align="left"> 3。 Dounce缓冲区</th></tr><tr><td> 10毫米</td><td>三</td><td> 0.242摹</td></tr><tr><td> 4毫米</td><td> MgCl 2的 </td><td> 0.163摹</td></tr><tr><td> 1毫米</td><td> 氯化钙 </td><td> 0.03摹</td></tr><tr><td colspan="3" align="center" style="padding:8px 0 16px 0">蒸压水，调节pH值至7.5和体积200毫升</td></tr></table>

neuronal nuclei isolation from human postmortem brain tissue

人类大脑中的神经元在很大程度上成为有丝分裂期间胎儿发育，从而保持其在整个全寿命核。然而，鲜为人知的是，在神经细胞染色质和核组织的发育和衰老过程中的变化，或在慢性神经精神疾病。然而，迄今为止最染色质和DNA检测（鱼除外）缺乏单细胞分辨率。为此，相当大的脑组织细胞的异质性带来明显的局限性，因为通常是不同的亚群的神经元与神经胶质细胞和其他非神经元细胞的不同类型混合的。一种可能的解决方案将增长细胞类型的特定的文化，但最中枢神经系统的细胞，包括神经细胞，体外可持续，充其量只有几个星期，从而将提供一个表观遗传机制的不完整的模型，可能整个全寿命。在这里，我们提供了一个协议冻结（从来没有固定）人死后脑细胞核提取和纯化。该方法包括低渗裂解液中提取的原子核，其次是超速离心法和反NeuN抗体immunotagging。标记神经元的细胞核，然后收集分别用荧光激活排序。这种方法应该适用于任何一个物种的广泛的大脑区域，适用于染色质免疫沉淀与现场和修改特异性抗组蛋白抗体，和DNA甲基化和其他实验研究。

Watch this Scientific Journal Video about Neuronal Nuclei Isolation from Human Postmortem Brain Tissue at JoVE.com

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

脑组织细胞的异质性，构成一个明显的局限性染色，因为大多数分析缺乏单细胞分辨率的表观遗传标记的研究。通常是神经元与神经胶质细胞和其他非神经元细胞混合。我们提供了一个协议提取和收集从人脑的神经元细胞核。

从人死后脑组织神经元细胞核分离

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Research

JoVE Journal

Biology

A Chromatin Assay for Human Brain Tissue

一个人脑组织中的染色质含量

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

科研

生物学

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

从小鼠胰腺RNA提取：核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Y-27632 丰富成人皮肤组织中人类黑色素细胞的产量

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

使用洗涤剂和酶无酶方法从整个组织进行核分离

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

使用基于多反应监测的人类脑组织蛋白质的多反应监测工作流程定量蛋白质组学工作流程

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...