Name: isolation and culture of rat embryonic neural cells: a quick protocol
Uploaded: 2012-05-24T12:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

We thank Jonna Ellis for editorial assistance. The project described was supported by Award Number R01MH079751 (PI: F. Peruzzi) from the National Institute of Mental Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institutes of Health.

1. Poly-D-Lysine (PDL): Preparation
<ol>
	<li>Add 5 ml of sterile ddH2O to 5 mg of PDL to obtain a stock solution of 1 mg/ml.</li>
	<li>Mix stock solution by pipetting several times.</li>
	<li>Use immediately or store Poly-D-Lysine Solution at 2-8 &deg;C.</li>
</ol>
2. Poly-D-Lysine (PDL): Coating Plastic Cell Culture Dishes
<ol>
	<li>Dilute the PDL stock solution with sterile ddH2O to the final concentration of 10 &mu;g/ml.</li>
	<li>Pipette enough solution into a 60 mm ...

<ol>
	<li>Aprea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubulin-mediated+binding+of+human+immunodeficiency+virus-1+Tat+to+the+cytoskeleton+causes+proteasomal-dependent+degradation+of+microtubule-associated+protein+2+and+neuronal+damage.">Tubulin-mediated binding of human immunodeficiency virus-1 Tat to the cytoskeleton causes proteasomal-dependent degradation of microtubule-associated protein 2 and neuronal damage.</a> J. Neurosci. 26, 4054-4062 (2006).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+rat+post-natal+and+fetal+brainstem+neurons.">Serum-free culture of rat post-natal and fetal brainstem neurons.</a> Brain Res. Dev. Brain Res. 120, 199-210 (2000).</li><li>Kivell, B. M., McDonald, F. J., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+serum-free+culture+of+late+fetal+and+early+postnatal+rat+brainstem+neurons.">Method for serum-free culture of late fetal and early postnatal rat brainstem neurons.</a> Brain Res. Brain Res. Protoc. 6, 91-99 (2001).</li><li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+B27/neurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum,+substantia+nigra,+septum,+cerebral+cortex,+cerebellum,+and+dentate+gyrus.">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li><li>Eletto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+SNAP25+expression+by+HIV-1+Tat+involves+the+activity+of+mir-128a.">Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a.</a> J. Cell Physiol. 216, 764-770 (2008).</li><li>Gualco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-IR-dependent+expression+of+Survivin+is+required+for+T-antigen-mediated+protection+from+apoptosis+and+proliferation+of+neural+progenitors.">IGF-IR-dependent expression of Survivin is required for T-antigen-mediated protection from apoptosis and proliferation of neural progenitors.</a> Cell Death Differ. 17, 439-451 (2010).</li><li>Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+differentiation+of+adult+neuronal+progenitor+cells+transplanted+to+the+adult+brain.">Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 11879-11883 (1995).</li><li>Keyser, D. O., Pellmar, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+in+the+hippocampus:+critical+role+for+glial+cells.">Synaptic transmission in the hippocampus: critical role for glial cells.</a> Glia. 10, 237-243 (1994).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 (1997).</li></ol>

The method of dissection and culture of rat hippocampal and cortical neurons described here allows performing experiments using nearly pure neuronal cultures grown in a chemically defined medium (Figure 3). Although protocols for culturing nearly pure neurons in serum-free media have been previously described2,3,4, there are important changes made in our method. Different from traditional protocols (i.e. Banker et al.)5, we have replaced trypsin with TrypLE Express, a more gentle di...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr>
 <td>Neurobasal</td>
 <td>98%</td>
 </tr>
 <tr>
 <td>B27</td>
 <td>2%</td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>0.5 mM</td>
 </tr>
 </tbody>
</table>
Table I. Neurobasal/B27 complete medium.
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr>
 <td>Glucose</td>
 <td>16 mM</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>22 mM</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>10 mM</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>160 mM</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>5 mM</td>
 </tr>
 <tr>
 <td>Na2HPO4</td>
 <td>1 mM</td>
 </tr>
 <tr>
 <td>KH2PO2</td>
 <td>0.22 mM</td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>50 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>250 ng/ml</td>
 </tr>
 <tr>
 <td>pH</td>
 <td>7.4</td>
 </tr>
 <tr>
 <td>Osmolarity</td>
 <td>320-330 mOsm</td>
 </tr>
 </tbody>
</table>
Table II. Dissection medium.
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Volume (&mu;l)</td>
 </tr>
 <tr>
 <td>Neurobasal/B27 complete medium</td>
 <td>240 </td>
 </tr>
 <tr>
 <td>Trypan Blue Stain 0.4%</td>
 <td>250</td>
 </tr>
 <tr>
 <td>Total</td>
 <td>490</td>
 </tr>
 </tbody>
</table>
Table III. 50x Counting solution.
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Cat. number</td>
 </tr>
 <tr>
 <td>Hibernate E</td>
 <td>Brainbits</td>
 <td>767171</td>
 </tr>
 <tr>
 <td>Neurobasal</td>
 <td>Gibco, Invitrogen</td>
 <td>21103-049</td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco, Invitrogen</td>
 <td>17504-044</td>
 </tr>
 <tr>
 <td>Fungizone</td>
 <td>Gibco, Invitrogen</td>
 <td>15290-018</td>
 </tr>
 <tr>
 <td>Gentamicin sulfate</td>
 <td>Sigma Aldrich</td>
 <td>G1264</td>
 </tr>
 <tr>
 <td>Glutamax 200 mM</td>
 <td>Gibco, Invitrogen</td>
 <td>35050</td>
 </tr>
 <tr>
 <td>TrypLE Express w/o phenol red</td>
 <td>Gibco, Invitrogen</td>
 <td>12604</td>
 </tr>
 <tr>
 <td>Cytosine-&beta;-D-arabinofuranoside hydrochloride</td>
 <td>Sigma Aldrich</td>
 <td>C6645</td>
 </tr>
 <tr>
 <td>Poly-D-Lysine</td>
 <td>Sigma Aldrich</td>
 <td>P6407</td>
 </tr>
 <tr>
 <td>Laminin 1 mg/ml</td>
 <td>Millipore</td>
 <td>CC095</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma Aldrich</td>
 <td>H3375</td>
 </tr>
 <tr>
 <td>Trypan Blue Stain 0.4%</td>
 <td>Gibco, Invitrogen</td>
 <td>15250</td>
 </tr>
 </tbody>
</table>
Table IV. Specific reagents.
<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Cat. number</td>
 </tr>
 <tr>
 <td>Stereo Microscope</td>
 <td>Olympus </td>
 <td>SZ61</td>
 </tr>
 <tr>
 <td>Large Forceps</td>
 <td>FST </td>
 <td>11022-14</td>
 </tr>
 <tr>
 <td>Fine-tipped forceps</td>
 <td>Moria</td>
 <td>MC40B</td>
 </tr>
 <tr>
 <td>Micro fine-tipped forceps</td>
 <td>Moria</td>
 <td>MC31</td>
 </tr>
 <tr>
 <td>Razor-sharp scissors</td>
 <td>Roboz</td>
 <td>RS-6820</td>
 </tr>
 <tr>
 <td>Micro Dissecting scissors</td>
 <td>FST </td>
 <td>91460-11</td>
 </tr>
 <tr>
 <td>Micro Dissecting Curved scissors</td>
 <td>FST </td>
 <td>14067-11</td>
 </tr>
 <tr>
 <td>Glass 2-chamber slides</td>
 <td>Lab-Tek</td>
 <td>154461</td>
 </tr>
 <tr>
 <td>60 mm dishes</td>
 <td>BD Falcon</td>
 <td>353002</td>
 </tr>
 <tr>
 <td>100 mm dishes</td>
 <td>Corning</td>
 <td>430167</td>
 </tr>
 <tr>
 <td>15 ml tubes</td>
 <td>BD Falcon</td>
 <td>352099</td>
 </tr>
 <tr>
 <td>1.5 ml cryo-tube vial</td>
 <td>Nunc</td>
 <td>375353</td>
 </tr>
 </tbody>
</table>
Table V. Specific equipment.

isolation and culture of rat embryonic neural cells: a quick protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

Watch this Scientific Journal Video about Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol at JoVE.com

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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