Name: preparation of dissociated mouse cortical neuron cultures
Uploaded: 2007-12-19T00:00:00
Description: Watch this Scientific Journal Video about Preparation of Dissociated Mouse Cortical Neuron Cultures at JoVE.com

<p class="jove_step">Preparations before day of culturing:</p>
<ul>
<li>Prepare sterile dissecting solution (DS).</li>
<li>Prepare NBM/B27 (Neurobasal Mediom with B27 supplements).</li>
<li>Autoclave ddH<sub>2</sub>O and sterilize glass coversilps, if needed.</li>
<li>Coat tissue culture dishes or glass coverslips with poly-D-lysine.</li>
</ul>
<p class="jove_step">Poly-D-Lysine Coating:</p>
<p class="jove_content">Prepare the day before culturing under sterile conditions.</p>
<ul>
<li>Thaw aliquot of 10X PDL and place on ice.</li>
<li>Add 9 ml...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5-Fluoro-2'-deoxyuridine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S9625</td>
<td>&nbsp;</td>
<tr>
<td>Vibraslicer</td>
<td>&nbsp;</td>
<td>Campden Instruments Ltd.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Potassim Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4504</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Phosphate Dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0876</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5379</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D (+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-Lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Media (NBM)</td>
<td>&nbsp;</td>
<td>Invirtogen (Gibco)</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-5-phosphonopentanoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5282</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9253</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Hydroxide, 1N solution</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SS266-1</td>
<td>&nbsp;</td>
<tr>
<td>L-Cysteine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7755</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0140-01</td>
<td>&nbsp;</td>
<tr>
<td>Papain</td>
<td>&nbsp;</td>
<td>Worthington </td>
<td>LS 03126</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 0.2 &micro;m Syringe Filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>DDA02025S0</td>
<td>&nbsp;</td>
<tr>
<td>Glass Coverslips, No1, &#216;12mm</td>
<td>&nbsp;</td>
<td>Bellco Glass Inc.</td>
<td>1943-00012</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Media (MEM)</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>11090-081</td>
<td>with Earle's salts
without L-glutamine, needed for growing non-neuronal cultures</td>
</tr>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>15070-063</td>
<td>for non-neuronal culture</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>16140-071</td>
<td>needed for non-neuronal cultures</td>
</tr>
<tr>
<td>Nunclon "Triple Flask" Tissue Culture Bottle</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-25</td>
<td>Use for non-neuronal cultures. these flasks are very convenient when producing "conditioned Neurobasal Media/B27", but any other tissue culture flasks or dishes can be used instead.</td>
</tr>
<tr>
<td>Glass bottom "Imaging dishes"</td>
<td>&nbsp;</td>
<td>MatTek Corp.</td>
<td>P35G-1.5-10-C</td>
<td>Glass bottomed, &#216;35mm culture dishes ideal for Calcium or Sodium Imaging when using an inverted imaging setup. But expensive!</td>
</tr>		</tbody>
		</table>
		</div>
	

preparation of dissociated mouse cortical neuron cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

Watch this Scientific Journal Video about Preparation of Dissociated Mouse Cortical Neuron Cultures at JoVE.com

Preparation of Dissociated Mouse Cortical Neuron Cultures

<p class="jove_content">This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.</p>

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Research

JoVE Journal

Biology

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Preparation of <em>Aplysia</em> Sensory-motor Neuronal Cell Cultures

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...