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

The authors appreciate the advice and technical support provided by Dr. Richard Konz and staff at the Flow Cytometry Core facility at the University of Massachusetts Medical School. Core resources supported by the Diabetes Endocrinology Research Center Grant DK032520 were also used. This work is supported by grants from the National Institute of Mental Health (NIMH), the National Institute of Drug Abuse (NIDA) and the National Institute of Child Health and Human Development.

Nuclei Extraction
<ol>
<li>Put&nbsp;250 mg of frozen postmortem brain tissue in 5 mL Lysis Buffer1 in a douncer and place it on ice. To receive about 5 million nuclei after FACS sorting, we usually use 1000 mg of tissue per sample.</li>
<li>Once the tissue has thawed, dounce the tissue for 1 minute while on ice.</li>
<li>Take the homogenized tissue and place it into a 15 mL clear ultracentrifuge tube. Put the tube on ice.</li>
<li>Take 9 mL of Sucrose Solution2 with a pipette, put the pipette at th...

<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>

The protocol presented here should be particularly useful for studies focused on epigenetic changes of neuronal chromatin and, more generally, on the molecular phenotype of the neuronal nucleus, during normal developing and aging, or in neurological or psychiatric disease. The method is of particular advantage when the cellular heterogeneity of brain tissue, including potential shifts in neuron-to-glia ration during the course of aging or due to disease are a concern 1. For example, by combining the present so...

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			<div>
			Reagents:

<table>
<tr><th colspan="3" align="left">1. Lysis Buffer</th></tr>
<tr><td>0.32M</td><td>Sucrose</td><td>5.47 g</td></tr>
<tr><td>5 mM</td><td>CaCl2</td><td>250 &micro;l</td></tr>
<tr><td>3 mM</td><td>Mg(Acetate)2</td><td>150 &micro;l</td></tr>
<tr><td>0.1 mM</td><td>EDTA</td><td>10 &micro;l</td></tr>
<tr><td>10mM</td><td>Tris-HCl, pH8</td><td> 500 &micro;l</td></tr>
<tr><td>1 mM </td><td>DTT</td><td>17 &micro;l</td></tr>
<tr><td>0.1%</td><td>Triton X-100</td><td>50 &micro;l</td></tr>
<tr><td colspan="3" align="center" style="padding:8px 0 16px 0">Adjust volume to 50 ml with Autoclaved water</td></tr>
</table>
<table>
<tr><th colspan="3" align="left">2. Sucrose Solution</th></tr>
<tr><td>1.8 M</td><td>Sucrose</td><td>30.78 g</td></tr>
<tr><td>3 mM </td><td>Mg(Acetate)2</td><td>150 &micro;l</td></tr>
<tr><td>1 mM</td><td>DTT</td><td>17 &micro;l</td></tr>
<tr><td>10 mM</td><td>Tris-HCl, pH8</td><td>500 &micro;l</td></tr>
<tr><td colspan="3" align="center" style="padding:8px 0 16px 0">Adjust volume to 50 ml with Autoclaved water</td></tr>
</table>
<table>
<tr><th colspan="3" align="left">3. Dounce buffer</th></tr>
<tr><td>10 mM</td><td>Tris</td><td>0.242 g</td></tr>
<tr><td>4 mM</td><td>MgCl2</td><td>0.163 g</td></tr>
<tr><td>1 mM</td><td>CaCl2</td><td>0.03 g</td></tr>
<tr><td colspan="3" align="center" style="padding:8px 0 16px 0">Adjust pH to 7.5 and volume to 200 ml with Autoclaved water</td></tr>
</table>		</div>

neuronal nuclei isolation from human postmortem brain tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

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

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

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

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.

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

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.

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.

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

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

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

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

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.

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.

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.

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).

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

Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nuclei RNA Sequencing

To begin, start the robotic dissociator. Turn on the cooling by setting the slider at the top of the right of the screen to Cool, and clicking on it to start cooling so that the slider appears orange. Next, check that the attached NSR and NIB bottles have sufficient liquid and are properly cooled.Remove the tissue samples from the minus 80 degrees Celsius freezer, and immediately place them on dry ice. Using a pre-cooled scalpel, cut the tissue into a 15 to 50 milligram piece on a pre-cooled Petri dish, or metal plate, placed on dry ice. Remove the nuclei isolation cartridge from the refrigerator and unpack it.Remove the grinder, and pipette 15 microliters of RNAs inhibitor to the bottom of the cartridge. Using tweezers, place the tissue sample at the bottom of the cartridge. Next, select Run a Protocol on the instrument, and click on the Nuclei option in the upper left corner.From the menu, select the Low Volume Nuclei isolation protocol, and then click on Modify to verify that the disruption speed is set to fastest. Then, open the instrument door. Insert the cartridge into the designated location.Rotate the cartridge lock, and slide into the stage until the red knob clicks into place. Close the door and click Next to start the nuclei extraction run. Once the run is finished, remove the cartridge from the instrument by lifting the red knob and pulling out the stage.Immediately place the cartridge on ice.

Purification of Nuclei from Mouse Brain for Single-Cell Multiome Analysis

To begin, fill a glass Dounce with two milliliters of ice-cold nuclei lysis buffer containing 0.01%digitonin, and add the dissected mouse brain tissue pieces into it. Using a glass Dounce tissue homogenizer, homogenize the tissue 25 times each with pestles A and B, and transfer the resulting homogenate into a 15-milliliter tube. Add two milliliters of ice-cold nuclei lysis buffer containing 0.01%digitonin to the homogenate and incubate on ice for five minutes.Then, centrifuge the nuclei at 500 G for five minutes at four degrees Celsius. Using a micropipette, remove the supernatant and add four milliliters of ice-cold nuclei lysis buffer containing 0.01%digitonin. Filter the nuclei suspension through a 40-micrometer cell strainer.Centrifuge the nuclei again, remove the supernatant and add four milliliters of staining buffer to wash the nuclei. After filtering the suspension through a 40-micrometer cell strainer, pellet the nuclei by centrifugation and resuspend the nuclei in one milliliter of PBS containing 0.04%BSA and RNase inhibitors. To count the cells, mix 10 microliters of 0.4%trypan blue with 10 microliters of the nuclei in a 0.5-milliliter tube.Count the nuclei using an automated cell counter. Transfer 100 microliters of the nuclei to a FACS tube for the unstained control. Add 10 microliters of 7-AAD to the remaining nuclei and incubate for five minutes at four degrees Celsius, and proceed with nuclei sorting using FACS.Then, transfer 10 microliters of the sorted nuclei into a new FACS tube containing 90 microliters of DPBS with 2%heat-inactivated FBS. After centrifuging the sorted nuclei, remove the supernatant using a micropipette and resuspend the nuclei in 100 microliters of diluted nuclei buffer. Before sorting, the sample contains substantial debris with over 99%of singlets positive for nuclear stain, indicating effective cell lysis.Brain nuclei after sorting demonstrated an increase in purity from the initial 36%to nearly 100%