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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation
<ol>
	<li>Brain tissue isolation 
	NOTE: Isolation should be performed on ice, using ice cold solutions.
	<ol>
		<li>Use newborn pups 1-3 days old for primary neonatal cell culture. 
		&#8203;NOTE: For the study presented here, Sprague Dawley hSOD1G93A and non-transgenic rats were used. For genotyping, rat tails were used for later DNA extraction and PCR.</li>
		<li>Sprinkle the pup&#39;s head with 70% ethanol and immediately decapitate it fast using scissors.</li>
		<li>Cut open the skin using small-angled scissors to expose the skull. Open the skull by making a cut from the foramen magnum toward the orbits. Next, make a perpendicular midline cut. Remove the brain from the skull and place it in a Petri dish containing phosphate-buffered saline (PBS). 
		NOTE: Clean the tools between pups to prevent cross-contamination between transgenic and non-transgenic pups. Isolation of the cortices from the rest of the brain should be done under a stereomicroscope.</li>
		<li>Using the tip of the forceps, tear the connections between both hemispheres. Then, separate the hemispheres with curved forceps by gently pushing a hemisphere from the center to the side.</li>
		<li>Remove the meninges by carefully tearing them with straight and curved forceps.</li>
		<li>Remove the hippocampus by pinching it with a curved forceps. Discard the hippocampus or use it for another cell culture preparation. 
		NOTE: Further steps should be performed under the laminar flow hood to ensure sterile conditions.</li>
	</ol>
	</li>
	<li>Tissue homogenization and dissociation
	<ol>
		<li>Transfer one cortex into a 15 mL tube filled with 3 mL of cold PBS. Pipet the suspension up and down with a 1 mL tip, completing 10-15 strokes until the suspension becomes homogeneous.&#8203;NOTE: Be very cautious not to produce bubbles in this process.</li>
		<li>Centrifuge at 500 &#215; g for 5 min. Remove the supernatant and resuspend the pellet in 3 mL of cold PBS by pipetting it up and down with a 1 mL tip. Repeat the centrifugation step. 
		NOTE: All solutions from this point should be prewarmed to 37 &#176;C.</li>
		<li>Discard the supernatant and resuspend the pellet in 2 mL of complete Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Transfer the homogenate to a 2 mL tube.</li>
		<li>Pass the homogenate through 21 G and 23 G needles (three times each) to make a suspension of single cells. 
		NOTE: Be very cautious not to produce bubbles in this process.</li>
	</ol>
	</li>
	<li>Pour the cell suspension prepared from one cortex into a 60 mm diameter Petri dish or a T25 flask (with the surface pretreated with a polypeptide coating for the growth of adherent cells) containing 3 mL of complete DMEM. Lightly shake the Petri dish so that the suspension is uniformly distributed.</li>
	<li>Grow the cells in the incubator in a humidified atmosphere of 5% CO2/95% air at 37 &#176;C.</li>
	<li>Change the medium 48 h after isolation and replace the medium every 3 days.</li>
	<li>To promote astrocyte growth, when cells reach 70%-80% confluency, wash them with prewarmed PBS to remove the loosely attached glial cells and traces of FBS in the medium.
	<ol>
		<li>Trypsinize the underlying layer of astrocytes by adding 1 mL of prewarmed trypsin solution (0.25% trypsin, 0.02% EDTA in PBS, sterile-filtered).</li>
		<li>Place the dish in the incubator at 37 &#176;C for 2-5 min.</li>
		<li>Check the cells under the microscope. When they start to detach, add 4 mL of complete DMEM.</li>
		<li>Collect the cell suspension and transfer it to a 15 mL tube. Centrifuge at 500 &#215; g for 5 min.</li>
		<li>Discard the supernatant and resuspend the pellet in 1 mL of complete medium. Count the cells using a hemocytometer.</li>
		<li>Replate the cells at a density of 104 cells/cm2 in 5 mL of fresh complete DMEM.</li>
	</ol>
	</li>
	<li>Change the medium every 3 days. To minimize the presence of other glial cell types, after astrocytes reach 50% confluence and prior to each media replacement, wash the cells with complete DMEM.
	<ol>
		<li>Aspirate the supernatant medium with a 1 mL pipette and gently dispense it onto the layer of cells several times. Ensure that the whole surface of the cell layer is covered during this washing step.</li>
	</ol>
	</li>
	<li>Once the cells reach 80% confluence (usually after 14 days), repeat the trypsinization and the collection of astrocytes as described in step 1.6.</li>
	<li>Seed 5 &#215; 103 of astrocytes on a 7 mm circular glass coverslip coated with poly-L-lysine (50 &#181;g/mL). Use them in experiments after 48 h.</li>
	<li>To promote microglia, after step 1.7, allow the glial cells to reach a confluent layer. When microglial cells appear on top of the astrocyte layer (after 10-15 days, recognized by their smaller, more oval bodies and shorter processes), shake the Petri dish on an orbital shaker for 2 h at 220 rpm.</li>
	<li>Dispersing and seeding microglial cells
	<ol>
		<li>Lightly wash the detached and loosely attached cells by aspirating the supernatant medium with a 1 mL pipette tip and gently dispense it onto the layer of cells several times. Be sure to cover the whole surface of the cell layer during this washing step, collect the medium with the detached cells, and transfer it to a 15 mL tube.</li>
		<li>Centrifuge at 500 &#215; g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of medium.</li>
		<li>Pass the cell suspension through a 21 G needle to obtain a single-cell suspension. 
		NOTE: Most published methods use trypsin or papain to dissociate the tissue; however, 21 G needles have the advantage of preventing the overdigestion of cells, allowing for a gentler dissociation.</li>
		<li>Count the cells using a hemocytometer. Seed 5 &#215; 103 microglial cells on a 7 mm circular glass coverslip coated with poly-L-lysine (50 &#181;g/mL). Use them in experiments after 48 h. 
		&#8203;NOTE: It is difficult to obtain a pure microglial culture by shaking, since the oligodendrocyte precursor cells and astrocytes may still be present in a variable number depending on the experimenter&#39;s experience.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mL tube</td>
			<td>Sarstedt, Germany</td>
			<td>72.691</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>Nipro, Japan</td>
			<td>HN-2138-ET</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G needle</td>
			<td>Nipro, Japan</td>
			<td>HN-2338-ET</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Nipro, Japan</td>
			<td>SY3-5SC-EC</td>
			<td></td>
		</tr>
		<tr>
			<td>6 mm circular glass coverslip</td>
			<td>Menzel Glasser, Germany</td>
			<td>630-2113</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>ThermoFisher Sientific, USA</td>
			<td>130181</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>EDS-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco, ThermoFisher Scientific, USA</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert GmbH + Co. KG, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carlo Erba Reagents, Spain</td>
			<td>471686</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin and Streptomycin</td>
			<td>ThermoFisher Sientific, USA</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a primary culture of astrocytes from a rat pup brain

Source: Mili&#263;evi&#263;, K., et al., <a href="https://app.jove.com/v/63483">Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.</a> J. Vis. Exp. (2022)
The video demonstrates a technique for culturing primary astrocytes from the rat cortex. The cortex is mechanically dissociated to obtain a homogeneous glial cell suspension, which is then plated on polypeptide-coated plates to promote selective proliferation of astrocytes. Subsequently, the astrocytes are enzymatically dissociated from the plates and transferred to fresh media, allowing the culture of pure astrocytes.

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Primary cell culture preparation

...

Add a cortex from a rat pup brain to a tube with a buffer.
With a pipette, gently move the cortex up and down to break it into small tissue pieces.
Centrifuge and discard the supernatant.
Resuspend the pieces in a culture medium.
Pass the pieces through needles of decreasing diameters to obtain a single-cell suspension.
Transfer the cells to a polypeptide-coated plate containing the culture medium and incubate.
Astrocytes strongly adhere to polypeptide-coated surfaces, facilitating their growth over other cells.
Remove the medium and wash with a buffer to remove any loosely attached cells.
Treat with a digestive enzyme to detach the astrocytes from the plate.
Add the medium to stop the enzymatic activity.
Collect the supernatant in a tube, and centrifuge.
Discard the supernatant and then resuspend the cells in a fresh culture medium.
Place the cells into a plate and incubate to facilitate the multiplication of astrocytes.

generating-a-primary-culture-of-astrocytes-from-a-rat-pup-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

222 조회수. 출처: Milićević, K., et al., 쥐 성상세포와 미세아교세포의 기본 배양 및 근위축성 측삭 경화증 연구에서의 사용. J. Vis. 특급 (2022) 이 비디오는 쥐의 피질에서 1차 성상세포를 배양하는 기술을 보여줍니다. 피질은 기계적으로 해리되어 균질한 신경교세포 현탁액을 얻은 다음 폴리펩타이드로 코팅된 플레이트에 도금되어 성상세포의 선택적 증식을 촉진합니다. 그 후, 성상세포는 ?...

비디오: 쥐 새끼 뇌에서 성상세포의 1차 배양 생성 - 개념

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

간접 신경-성상 세포 공동 배양 분석 일 :<em&gt; 체외</em&gt; 신경 세포 - glia 상호 작용의 상세한 조사를위한 셋업

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

연구

JoVE Journal

신경과학

Live-imaging of Mitochondrial System in Cultured Astrocytes

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and function in astrocytes are implicated in cognition. This article describes the method for time-lapse imaging of astrocyte cultures equipped with a mitochondrial biosensor: MitoTimer. MitoTimer is a powerful and unique tool to assess mitochondrial dynamics, mobility, morphology, biogenesis, and redox state. Here, the different procedures for culture, image acquisitions, and subsequent mitochondrial analysis are presented.

This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.

배양 된 성상 세포에서 미토콘드리아 시스템의 라이브 이미징

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

마우스 성상세포의 분리 및 직접 뉴런 리프로그래밍

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

Generating a Primary Culture of Astrocytes from a Rat Pup Brain

내레이션 대본

Generating a Primary Culture of Astrocytes from a Rat Pup Brain