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, ...

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