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蔗糖溶液与蔗糖溶液 2顶部的匀浆组织的解决方案，以创建一个浓度梯度。 </li><li>权衡超速离心机管，以确保内超速离心机旋转时，他们都将平衡。加入裂解液1至顶层的任何重量的调整。 </li><li>管已称量后，放入转子的水桶。 </li><li>放入一个SW28转子所有的水桶，小心地放入超速离心机的转子。 </li><li>超速离心机样品107163.6离心力为2.5小时，4 ° C。 </li><li>一旦完成离心，去除上清液，同时发现在浓度梯度的碎片层，使用真空，同时小心不要打扰颗粒包含的原子核在试管底部。 </li><li>每个颗粒，并添加500μL的1X PBS让冰坐了20分钟。这使得颗粒溶于自己的一点点，使得更容易稍后机械解散移液器向上和向下，这减少的压力量的原子核经验。 </li><li>孵育冰20分钟后，原子核，机械移液器上下完全溶解细胞核内1XPBS。 </li><li>从超速离心机管取出的原子核包含的解决方案，并结合成一个单一的2 ml离心管，两管的内容。同样，放在冰的样品。 </li></ol>免疫染色为流式细胞仪<ol><li>在这一点上，使免疫中小学抗体组成的混合物，并阻止混合。对于每个样品，300μL含1.2μLNeuN抗体，100μL阻塞组合，其中包含0.5％BSA和10％正常山羊血清，在1XPBS和1μL的Alexa Fluor 488 1XPBS结合起来。对照样本中，排除的主要抗体NeuN，相反，只需添加更多1XPBS。 </li><li>涡免疫混合物和孵化5分钟，在室温下在黑暗中。 </li><li>虽然染色的混合物，是培育，使样品的控制。排序，样品，仅含有核的流式细胞仪，称为“不染控制”是必要的;第二个样品，阴性对照，含二级抗体的Alexa Fluor 488，无主的抗体NeuN，还需要原子核。 </li><li>分装20μL的解决方案包含到另一个2 mL含980 UL阴性对照1XPBS管1XPBS不染控制，和另外20微升2 ml离心管核。提高卷的原子核包含示例备份1XPBS至1 mL。所有样品放置在冰上。 </li><li>免疫混合物现在已完成孵化，其内容添加到相应的样品401μL。原子核的样本将收到NeuN的Alexa Fluor 488的混合物，而阴性样品将收到含有二级抗体的Alexa Fluor 488的混合物。 </li><li>考虑到冷藏室的样品，在黑暗中旋转了45分钟。 </li><li>样品在黑暗中孵育45分钟后，置于冰上，并给他们带来的流式细胞仪设施。在流式细胞仪设施的运输，样品应保持在冰上，在黑暗中。 </li></ol>流式细胞仪<ol><li>在流式细胞仪的工厂，通过一个40微米的过滤器过滤的样品。然后，运行通过了几分钟的工具，同时又保持低温，以排序前得到一些初步的数据。 </li><li>在这一点上，它是必要的设置进行排序所需的适当的盖茨。几种不同的门是用来排序的样品。第一门提供了一个样本内发现了不同的原子核的大小的想法，并允许从实际细胞核碎片separatation。第二个门，让我们单独排序原子核。丢弃任何聚合被丢弃在这一点上。最后，第三个门是设置一个特定的荧光波长，匹配的荧光，发现在我们的第二抗体。配售这扇大门，使我们能够进行排序和单独NeuN +，因此从NeuN神经核 - 核。 </li><li>所有的门都选择后，原子核可以分为1XPBS。 </li><li>一旦经历了整个样本，检查通过仪器运行的有序样品等分纯度的排序。确保样品的荧光是不扩散，而是包含在一个单一的象限。这是最好的选择样本超过90％的纯。 </li></ol>后，流式细胞仪<ol><li>一旦样品已排序，处理的细胞核，就可以开始。排序样品取10毫升，以及加入2毫升的蔗糖溶液 2，有50 UL 1M氯化钙和30 UL 1M毫克（ACE） 2。在此提醒您，永远是因为原子核是不固定的的，一定要保持冰样本。 </li><li>如果有小于10毫升排序样本，提高音量至10 mL，加入1XPBS，或调整相应的补充材料。 </li><li> 15分钟，反转样品几次冰和地点。 </li><li>孵育15分钟，冰的样品后，在Swing桶转子离心15分钟4，在1786 离心力° C 。 </li><li>倒掉上清，与使用的真空，而不会干扰在试管底部发现白色颗粒。 </li><li>沉淀溶解于200μL的任何解决方案，您需要为出发实验，吸管向上和向下。 </li><li>解决方案，并转移到一个较小的管放入-80℃冷冻，直到进一步利用。 </li></ol>

<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, ...

neuronal-nuclei-isolation-from-human-postmortem-brain-tissue

Research

JoVE Journal

Biology

22.0K 次观看.  University of Massachusetts Medical School. 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。

视频: Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

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

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

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

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

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 ...

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

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

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 ...

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 executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

从成年小鼠肾脏中分离细胞核以进行单核RNA测序

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

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

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 ...

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

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

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 ...

从冷冻哺乳动物组织中分离和分析单个细胞核

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

作者聚焦：增强药物发现 - 开发从冷冻组织中提取细胞核的自动化标准化方案 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.