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.
Nuclei Extraction
Immunostaining for FACS
FACS
After FACS
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 sorting protocol with chromatin immunoprecipitation techniques, we recently described neuron-specific histone methylation patterns at the brain derived neurotrophic factor (BDNF) gene promoter, and for additional neuronal genes 2. A similar method had been used previously to collect neurons from adult cerebral cortex for retrospective birth dating 3. The sorting technique was also employed in mouse brain 2, and because of the stability of nuclei in frozen-then-thawed tissue, we anticipate that the approach presented here is applicable to a wide range of species. Because chromatin immunoprecipitation from as little as 10,000 cells for is now feasible even for a more comprehensive genome coverage 4, it may be possible to use much less than the 1000 mg of starting material that we recommended above.
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.
Reagents:
1. Lysis Buffer | ||
---|---|---|
0.32M | Sucrose | 5.47 g |
5 mM | CaCl2 | 250 µl |
3 mM | Mg(Acetate)2 | 150 µl |
0.1 mM | EDTA | 10 µl |
10mM | Tris-HCl, pH8 | 500 µl |
1 mM | DTT | 17 µl |
0.1% | Triton X-100 | 50 µl |
Adjust volume to 50 ml with Autoclaved water |
2. Sucrose Solution | ||
---|---|---|
1.8 M | Sucrose | 30.78 g |
3 mM | Mg(Acetate)2 | 150 µl |
1 mM | DTT | 17 µl |
10 mM | Tris-HCl, pH8 | 500 µl |
Adjust volume to 50 ml with Autoclaved water |
3. Dounce buffer | ||
---|---|---|
10 mM | Tris | 0.242 g |
4 mM | MgCl2 | 0.163 g |
1 mM | CaCl2 | 0.03 g |
Adjust pH to 7.5 and volume to 200 ml with Autoclaved water |