eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Dissociation of Ventral Midbrain Cells
<ol>
	<li>Transfer the pieces of the ventral midbrain under a laminar flow hood. Remove the Hanks&#39; balanced salt solution (HBSS) and add 1 ml pre-warmed (37 &#186;C) 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) into the conical tube (volumes enough for up to 12 pieces of ventral midbrain). Incubate the tissue at 37 &#186;C for 5-10 min.</li>
	<li>Remove trypsin-EDTA under the hood and add 1 ml de-activation medium (the serum will deactivate trypsin) to the tissue.</li>
	<li>Remove the deactivation medium and wash the tissue in 1 ml of the complete medium twice. 
	NOTE: To prevent discarding the dissociated cells, avoid removing all the medium from the tube and pipetting the tissue.</li>
	<li>Add 1 ml of complete medium and triturate the tissue with a fire-polished glass pipette. Avoid bubble formation and continue triturating until a single-cell suspension is achieved. 
	NOTE: Usually, 8-10 passes through the restricted tip are required for complete dissociation.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at room temperature (RT) and remove the medium.</li>
	<li>Re-suspend the pellet in 1 ml complete medium by slowly pipetting up and down up to four times.</li>
</ol>
2. Plating Ventral Midbrain Cells
<ol>
	<li>Mix 10 &#181;l of the cell suspension with 90 &#181;l Trypan blue solution and count the number of cells with a hemocytometer. 
	NOTE: The viability of the cells can also be checked at this stage.</li>
	<li>Place sterilized microcentrifuge tube caps in 100 mm Petri dishes and transfer the Poly-L-ornithine/Laminin coated coverslips, using forceps, one at a time on top of the caps. 
	NOTE: Do not wash the coverslips and avoid drying the laminin, as this may cause uneven cultures and decrease the cells&#39; survivability.</li>
	<li>Adjust the volume to 1,500 cells per 1 &#181;l by adding a complete medium and 100 &#181;l (total of 150,000 cells) of the cell suspension on top of each coverslip (the volume is optimal to cover the surface of the coverslip without spillage). Close the Petri dishes and incubate them for 1 hr in a humidified tissue culture incubator (37 &#176;C, 5% CO2). 
	NOTE: CRITICAL STEP: This step is required to enhance the attachment and viability of the cells and to increase the number of coverslips per embryo. Alternatively, the cells can be transferred directly into the wells (containing coverslips), but the number of cells should be increased to 350,000 per well (instead of 150,000). In other words, using this method, 8-10 coverslips can be acquired, while direct transfer yields about 3-4 coverslips because of the cells that attach to the plates (beside or underneath the coverslips). The average viability of the cultures using this method was around 90%.</li>
</ol>
3. Culture Growth and Maintenance
<ol>
	<li>After 1 hr of incubation, carefully transfer the coverslips, including the medium, into 24-well plates containing 400 &#181;l pre-warmed (to 37 &#186;C) complete medium (total volume: 500 &#181;l). Incubate the cells overnight (O/N) at 37 &#186;C.</li>
	<li>Within 24 hr after incubation of the wells, gently add 500 &#181;l complete medium into each well. 
	NOTE: The first few hours are the most critical stage for the survival of the dopaminergic neurons, and the number of surviving cells does not change significantly beyond this point.</li>
	<li>Do not add additional media to the cultures for the first two weeks or until the medium becomes yellow. If culturing for more than two weeks or the medium color changes to yellow, exchange half of the media (500 &#181;l) with fresh, complete pre-warmed media (typically every two weeks). 
	NOTE: Dopaminergic neurons within the cultures would survive for more than six weeks under these conditions.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium nutrient mixture F-12</td>
			<td>Invitrogen</td>
			<td>11330</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td>Invitrogen</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, heatinactivated (FBS)</td>
			<td>Invitrogen</td>
			<td>16140</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement (100X), liquid</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution (45% (wt/ vol) in water</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin BSA&#160;</td>
			<td>Sigma</td>
			<td>A9430</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin from Engelbreth-HolmSwarm murine sarcoma basement membrane&#160;</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Invitrogen</td>
			<td>15070</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (0.05% (wt/vol)</td>
			<td>Invitrogen</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) cell culture tested</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine, 0.01% solution</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution (0.4% (wt/vol))</td>
			<td>Biowhittaker</td>
			<td>17-942E</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing and maintaining dopaminergic neurons from mouse embryonic brain tissue

Source: Weinert, M. et al., <a href="https://app.jove.com/v/52475">Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains.</a> J. Vis. Exp. (2015)
This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Treat the embryonic midbrain tissue, rich in developing dopaminergic neurons, with a trypsin-EDTA solution to detach them from the tissue matrix.
Add a deactivation medium to inhibit enzyme activity, and then wash to remove residual enzymes.
Pipette the tissue repeatedly. The embryonic neurons possess fewer projections, reducing the stress during this disturbance and generating a single-cell suspension.
Centrifuge and remove the supernatant, then resuspend the neurons in a complete medium. Transfer these neurons onto a polymer-coated coverslip for cell attachment.
Place the coverslip in a multi-well plate with a complete medium, providing nutrients and growth factors essential for neuron growth.
The antibiotics in the medium prevent bacterial growth, supporting neuronal viability.
As neurons grow and mature, they develop cellular projections, such as axons and dendrites, forming synaptic connections.
Periodically, remove half of the used media to eliminate toxic by-products.
Add a fresh medium for the long-term maintenance of the dopaminergic neurons.

culturing-maintaining-dopaminergic-neurons-from-mouse-embryonic-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

45 vistas. Fuente: Weinert, M. et al., Aislamiento, cultivo y mantenimiento a largo plazo de neuronas dopaminérgicas mesencefálicas primarias a partir de cerebros de roedores embrionarios. J. Vis. Exp. (2015) Este video describe un método para cultivar neuronas dopaminérgicas a partir de tejido cerebral embrionario de ratón. El proceso incluye el tratamiento del mesencéfalo con una enzima proteolítica, la separación de las células y el cultivo de las mismas en cubreobjetos recubiertos de polímer...

Video: Cultivo y mantenimiento de neuronas dopaminérgicas a partir de tejido cerebral embrionario de ratón - Concepto

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Un método simplificado para determinar la densidad ultra-baja, a largo plazo del hipocampo primaria Neurona Cultura

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Investigación

JoVE Journal

Neurociencias

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultivo primario de neuronas aisladas del cerebelo del ratón embrionario

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Aislamiento y cultivo de las neuronas motoras oculomotoras, trocleas y espinales de Islmnprenatal :GFP Ratones transgénicos

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

Transcripción

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue