eoe sub articles

EoE Sub Articles

1. 24-well Plates Preparation for High-Density Neuron Cultures
NOTE: Perform the following steps (2-3) the day before the planned harvest of embryos.
<ol>
	<li>Bring two 24-well plates into the cell culture hood and open the individual packaging.</li>
	<li>Connect an 18 G syringe needle to a 1 ml plastic single-use syringe.</li>
	<li>Hold the syringe and aim the needle tip at ~1 mm left to the center of the bottom of the well. Apply moderate force (~250 g) to etch the bottom of the well (~1 mm long). A groove will be formed, and the plastic materials displaced will form elevated support above the flat bottom well surface. Etch another ~1 mm-long parallel groove at ~1 mm to the right of the center of the bottom in a similar way.</li>
	<li>Repeat this step for all the wells of the two 24-well plates. The two 24-well plates used for high-density culture are now ready for coating with poly-D-lysine (see step 2.8 below).</li>
</ol>
2. Coverslips Preparation for Low-Density Neuron Cultures
<ol>
	<li>Use 12-mm diameter #1 round glass.</li>
	<li>Place 50 coverslips inside a 100 mm diameter sterile plastic petri dish and submerge coverslips in 20 ml 70% ethanol solution. Place this petri dish on a rotating platform with moderate (70 rpm) speed rotation for an hour. Remove 70% ethanol and then replace with a fresh batch of 70% ethanol. Rotate and wash for another hour.</li>
	<li>Discard 70% ethanol cleaning solution. Add sterile water (filtered through 0.2 &#181;m filter unit). Rinse the coverslips rigorously by rotating the petri dish by hand. Rinse three times, each time replacing with fresh sterile water.</li>
	<li>Place the petri dish with coverslips inside a sterile cell culture hood with laminar flow. Aspirate the rinsing water through the vacuum line and add 10 ml filtered sterile water to immerse the coverslips. The coverslips should be sterile, and the surface should be clean enough for poly-D-lysine coating.</li>
	<li>Open two 24-well plates from their individual packing and place them in the hood.</li>
	<li>Turn on the vacuum line in the hood. Connect a 200 &#181;l pipette tip to the vacuum line and use a scissor to cut the tip to create a larger opening.</li>
	<li>Use a fine-tip #5 tweezers to pick up coverslips from the petri dish, one at a time, and use the vacuum line to completely dry the coverslip. Then place one coverslip into each of the wells of the 24-well plate. The 50 cleaned coverslips should be enough to fill each of the wells of the two 24-well plates.</li>
	<li>Dilute the poly-D-lysine stock solution (1 mg/ml) into a working solution (0.1 mg/ml) with a borate buffer (pH 8.5). For the four 24-well plates (including two high density plates with etched bottom), prepare 30 ml of total working solution.</li>
	<li>Add 300 &#181;l 0.1 mg/ml poly-D-lysine work solution to each well with glass coverslips. Make sure the coverslips are completely immersed in solution. If not, use the #5 tweezer tip to press down coverslips against the bottom of the well, and use the tip to guide poly-D-lysine solution to cover all the surface of the coverslips. Visually inspect all the coverslips to make sure no air was trapped between the plate and coverslips.</li>
	<li>Add 300 &#181;l 0.1 mg/ml poly-D-lysine work solution to each well of the high-density culture plate with etched bottom. Rotate by hand to spread the solution to cover the entire bottom of the well.</li>
	<li>Return all the four plates with poly-D-lysine to the culture incubator, and incubate O/N.</li>
</ol>
3. Washing and Pre-conditioning Plates for Culture
NOTE: The following steps (3-5) are carried out on the day of tissue harvest.
<ol>
	<li>After incubation/coating of coverslips and 24-well plates O/N, bring all four plates (two with donor coverslips, two acceptor high density plates with etched plastic bottom) into the culture hood. Use the vacuum line to remove the poly-D-lysine solution.</li>
	<li>Immediately following the removal of the poly-D-lysine solution, add ~1 ml sterile water to each well using a plastic transfer pipette. After all the wells are filled with water, let stand for 10 min. Remove water by vacuum, then add 300 &#181;l Wash medium in each well to cover the bottom of the well (with or without glass coverslips). Do not let the poly-D-lysine coating dry out.</li>
	<li>Return all four plates to the cell incubator. The plates are ready to use once the dispersed hippocampal neurons are prepared.</li>
</ol>
4. Plating of Neurons and Long-Term Co-culture
<ol>
	<li>Bring the two 24-well plates with etched bottoms for high density neurons into the cell culture hood. Remove the 300 &#181;l precondition medium by vacuum. Plate 0.6 ml high density cell suspensions at 250,000 neurons/ml.</li>
	<li>Bring the other two 24-well plates with coverslips into the culture hood and remove the 300 &#181;l precondition medium by vacuum. Plate 0.6 ml low density cell suspensions at 10,000 neurons/ml on the coverslips inside the two 24-well plates.</li>
	<li>Return all four 24-well plates to the incubator. Let sit for 2 hr.</li>
	<li>Flip the low-density coverslips with adhering neurons to the high-density culture. Make sure the neurons are facing each other (i.e., not separated by the glass coverslip). Then return the two plates with co-culture to the incubator.</li>
</ol>
5. Co-culture Sustaining
<ol>
	<li>Feed co-cultures by adding 300 &#181;l fresh Feed medium (see Materials) at day&#160;in vitro&#160;(DIV) 5. There is no need to remove 50% of the original complete culture medium, as reported by many other studies. The feeding medium also contains 15 &#181;M cytosine arabinoside (Ara-C, to yield a final concentration of 5 &#181;M) to minimize the chance of glia contamination and proliferation.</li>
	<li>Following this initial feeding, add 300 &#181;l fresh Feed medium without Ara-C to the well every week. Should the culture be kept for more than one month, replace half of the medium with fresh Feed medium every week beyond one month time point (with the first medium half-replacement at DIV33). Morphologically, both high density and low-density neurons look healthy up to three months (the longest time observed by the experimenters).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Aliquot, store in 0.6ml size</td>
		</tr>
		<tr>
			<td>GlutaMAX-I</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>Dilute 100X&#160;</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic (AA)</td>
			<td>Life Technologies</td>
			<td>15240-096</td>
			<td>Dilute 100X&#160;</td>
		</tr>
		<tr>
			<td>Complete Culture medium</td>
			<td></td>
			<td></td>
			<td>Neurobasal medium with 1X B27, 1X AA, 1X GlutaMAX-I</td>
		</tr>
		<tr>
			<td>Wash medium</td>
			<td></td>
			<td></td>
			<td>same as &#39;Neurobasal medium&#39;</td>
		</tr>
		<tr>
			<td>Feed medium</td>
			<td></td>
			<td></td>
			<td>Neurobasal with 1X B27 supplement</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>M.W. 70000-150000</td>
		</tr>
		<tr>
			<td>Borate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>B6768 (boric acid); 71997(borax)</td>
			<td>1.24g boric acid &#38; 1.9g borax in 400ml H2O, pH to 8.5 use HCl</td>
		</tr>
		<tr>
			<td>12-mm round glass coverslips</td>
			<td>Glasswarenfabrik Karl Hecht GmbH</td>
			<td>1001/12</td>
			<td>No. 1 glass, purchase from Carolina Biological Supply</td>
		</tr>
	</tbody>
</table>

generating an ultra-low-density neuronal culture using a high-density neuronal feeder layer

Source: Lu, Z. et al., <a href="https://app.jove.com/v/53797">A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.</a>&#160;J. Vis. Exp.&#160;(2016).
This video demonstrates the method for culturing ultra-low-density neurons in the presence of a high-density neuronal feeder layer. It establishes a co-culture of varying-density neurons, ensuring close physical proximity. The growth factors secreted by high-density neurons help neuronal survival and growth, maintaining ultra-low-density neurons for a longer time period.

Generating an Ultra-Low-Density Neuronal Culture Using a High-Density Neuronal Feeder Layer

1. 24-well Plates Preparation for High-Density Neuron Cultures
NOTE: Perform the following steps (2-3) the day before the planned harvest of embryos.

	...

Begin with two multi-well plates: one with an etched bottom coated with a polymer and the other featuring a polymer-coated coverslip.&#160;
Seed a high-density suspension of the embryonic neurons into the plate with the etched bottom. Add an ultra-low-density neuron suspension to the coverslip containing well.
Incubate to allow neuronal attachment to the polymers.
Remove the coverslip and place it over the high-density neuron culture, enabling the neurons to face each other.
The etched surface with elevated structures creates a micro-space that separates neurons of different densities.
The high-density culture acts as a feeder layer and secretes the neural growth factor.
Due to the confined diffusion space, the growth factors accumulate around the neurons, facilitating their growth and the development of neuronal projections.
Introduce a neurobasal medium with cytosine arabinoside that selectively inhibits the growth of the non-neuronal cells.
Regularly replenish with a fresh neurobasal medium to maintain the neuronal culture.

generating-an-ultra-low-density-neuronal-culture-using-high-density

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

59 visualizações. Fonte: Lu, Z. et al., Um método simplificado para cultura de neurônios primários do hipocampo de densidade ultrabaixa e longo prazo.J. Vis. Exp.(2016). Este vídeo demonstra o método para cultivar neurônios de densidade ultrabaixa na presença de uma camada alimentadora neuronal de alta densidade. Ele estabelece uma co-cultura de neurônios de densidade variável, garantindo proximidade física. Os fatores de crescimento secretados pelos neurônios de alta densidade ajudam na sobrevivênci...

Vídeo: Gerando uma cultura neuronal de densidade ultrabaixa usando uma camada alimentadora neuronal de alta densidade - Conceito

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Low-Density hipocampo Cultura Neuron primária

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

JoVE Journal

Neurociência

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Uso de neurônios hipocampais primários para estudar a montagem de segmentos iniciais de Axon

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

Cultura Primária de Baixa Densidade Fácil e Reprodutível usando Estoque Congelado de Neurônios Embrionários do Hipocampo

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

Generating an Ultra-Low-Density Neuronal Culture Using a High-Density Neuronal Feeder Layer

Transcrição

Generating an Ultra-Low-Density Neuronal Culture Using a High-Density Neuronal Feeder Layer