isolation and culture of primary embryonic mouse midbrain dopamine neurons

Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons

To set up primary embryonic midbrain cultures from mouse embryonic day 13.5 mouse embryos, pool all of the harvested mid-brain floors into the same 1.5-milliliter tube, and wash the samples three times, with 500 microliters of calcium and magnesium-free HBSS per wash. After the last wash, replace the HBSS with 0.5% trypsin for a 30-minute incubation at 37 degrees Celsius.
At the end of the incubation, add 500 microliters of a freshly prepared DNase I in FBS solution to the partially digested tissue and use a siliconized glass pipette with a fire-polished tip to triturate the tissue. When only tiny, barely visible particles can be observed, allow these tissue fragments to settle to the bottom of the microcentrifuge tube, and transfer the supernatant into an empty 15-milliliter conical polypropylene tube.
Add one milliliter of HBSS to the tube containing DNase I in FBS, and mix several times by pipetting. Transfer one milliliter of this solution to the remaining tissue particles, and triturate the samples again. Then, pool the digested cell suspension with the tube of supernatant without transferring any remaining tissue fragments. After triturating the tissue sample one more time with the remaining DNase I in FBS in HBS, sediment the collected cells by centrifugation, and aspirate the supernatant without disturbing the pellet.
Wash the cells two times in two milliliters of warmed dopamine neuron medium per wash, and resuspend the cells at 3 x 104 cells per six microliters of fresh, warm medium concentration in a microcentrifuge tube. Next, remove the medium from each previously prepared micro-island and mix the cells with gentle pipetting before using a 10-microliter pipette to add six microliters of cells to each micro-island.
When all of the cells have been added, fill the empty wells at the edges of the plate with 150 microliters of water or PBS, and place the plate in the cell culture incubator for one hour. At the end of the incubation, add 100 microliters of fresh dopamine neuron medium to each well, and return the plate to the incubator.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation 
<ol>
	<li>Prepare dopamine neuron medium (DPM) with 0.46% D-glucose, 1% L-glutamine, 1% N2, 0.2% primocin, completed with Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F12). Filter the DPM after mixing the ingredients. Store DPM at 4 &#176;C and warm each aliquot only once. 
	NOTE: DPM should not contain Glial cell line-derived growth factor (GDNF), as it will reduce &#945;-synuclein accumulation in dopamine neurons.</li>
	<li>Prepare siliconized glass pipettes that are extremely hydrophobic, thereby minimizing the attachment to the surface and loss of cells during the initial handling of embryonic neurons.
	<ol>
		<li>Add 10 mL of siliconizing fluid to 1 L of distilled water and mix by stirring in a 2 L vessel. Leave the glass pipettes immersed in the siliconizing solution for 15 min.</li>
		<li>Rinse the pipettes 3&#8211;5x with distilled water. Dry the pipettes overnight at room temperature (RT) or for 1&#8211; 2 h at 100-120 &#176;C heated sterile space to speed up the drying.</li>
		<li>Sterilize the pipettes by standard autoclaving in a sealed autoclave bag.</li>
	</ol>
	</li>
	<li>Prepare poly-L-ornithine (PO) coated 96 well plates with transparent bottoms by adding 60 &#956;L of PO solution into the middle wells of the 96 well plate to be used for seeding of the neurons, leaving at least one row/column of wells at the edges of the plate to avoid edge effects. Keep the coated plate overnight at 4 &#176;C or 4 h at RT.</li>
	<li>Prior to plating the cells, aspirate PO completely and wash the cells thrice with 100 &#956;L of 1x phosphate-buffered saline (PBS). Aspirate 1x PBS from the wells and keep the lid of the plate open for complete drying. 
	NOTE: It is possible to collect used PO and filter it for reusing. This can be repeated twice for the same PO solution.</li>
	<li>Add 50 &#956;L of DPM to previously coated wells. Aspirate DPM from the wells with a 100 &#956;L plastic tip and simultaneously scratch the bottom of the well with circular movements to remove the coating at the perimeter of each well. A PO-coated island will remain in the middle of the well.</li>
	<li>Under a laminar hood, add 10 &#956;L of DPM to the middle of each coated island to create micro islands. 
	NOTE: A plate with DPM-covered micro islands can be kept under the laminar flow hood for 1&#8211;2 h during the isolation of cells</li>
</ol>
2. Isolation of the ventral midbrain floor from embryonic day 13.5 (E13.5) mouse embryos 
<ol>
	<li>Prior to dissection, fill a 10 cm Petri dish with Dulbecco&#39;s buffer and keep it on ice.</li>
	<li>Euthanize a E13.5 pregnant female mouse according to the institution&#39;s guidelines. Place the mouse flat on its back and spray the anterior body with 70% ethanol. Lift the skin above the womb with forceps and make an incision with surgical scissors to expose the uterus.</li>
	<li>Carefully remove the uterus and place it into the previously prepared Petri dish on ice.</li>
	<li>Using surgical scissors under the laminar hood at RT, carefully remove the embryos from the uterus. Remove all placental residue from the embryos with forceps and place them into a new 10 cm Petri dish filled with Dulbecco&#39;s buffer.</li>
	<li>Using dissection forceps or needles, cut off the hindquarter of the head from the places marked with black arrows in Figure 1A. Take the cut piece away from the rest of the embryo (Figure 1B).</li>
	<li>Place the posterior of the cut piece towards the observer (Figure 1C) and gently cut it open from caudal to cranial (Figure 1D). From 0.5 mm below the cranial opening, cut a 2 mm2 &#8211;3 mm2 region, shown in Figure 1E.</li>
	<li>Collect the ventral midbrain floor (see Figure 1F) in an empty 1.5 mL microcentrifuge tube. Keep the microcentrifuge tube on ice until all midbrain floors are collected in it. 
	NOTE:&#160;Alternatively, the midbrain floors can be collected with a 1 mL micropipette after dissection of all embryo brains.</li>
</ol>
3. Establishing primary embryonic midbrain cultures from E13.5 mouse embryos in 96 well plate format
<ol>
	<li>After the collection of midbrain floors from all embryos in the same 1.5 mL tube, remove the residual Dulbecco&#39;s buffer and wash the tissue pieces thrice with 500 &#956;L of Ca2+, Mg2+-free Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Remove HBSS and add 500 &#956;L of 0.5% trypsin to the tube. Incubate it at 37 &#176;C for 30 min.</li>
	<li>During incubation, warm 1.5 mL of fetal bovine serum (FBS) at 37 &#176;C, add 30 &#956;L of DNase I to the FBS, and mix. Also, fire-polish the tip of a siliconized glass pipette. Make sure that the hole has no sharp edges and is around the same size as a 1 mL micropipette tip. 
	NOTE: As an alternative, a low adhesion 1 mL micropipette tip can be used for trituration. However, siliconized glass pipettes seem to give the best results.</li>
	<li>As soon as the incubation in step 3.2 ends, add 500 &#956;L of the FBS/DNase mix to the partially digested tissue. Use the glass pipette to triturate the tissue in the FBS/ trypsin mix. Triturate until tissues dissociate into tiny, barely visible particles. Avoid bubbles during trituration.</li>
	<li>Let the leftover particles precipitate at the bottom of the microcentrifuge tube by gravity. Without pipetting the precipitate at the bottom, collect the supernatant into an empty 15 mL conical polypropylene tube.</li>
	<li>Dilute FBS/DNase I from step 3.3 (98:2) with 1,000 &#956;L of HBSS to obtain FBS/DNase-I/HBSS (49:1:50). Mix by pipetting up and down. Add 1,000 &#956;L of the new mix to the leftover particles in the microcentrifuge tube. Triturate again and repeat step 3.5.</li>
	<li>Repeat the previous step once more to use up all FBS/ DNase-I/HBSS (49:1:50).</li>
	<li>Once all the supernatant is collected inside the 15 mL tube (from steps 3.5, 3.7, and 3.8), use a tabletop centrifuge to spin down the supernatant (~3 mL) at 100 x g, for 5 min. Remove the supernatant without touching the pelleted cells at the bottom.</li>
	<li>Wash the cell pellet by adding 2 mL of DPM to the tube and spin it down at 100 x g for 5 min. Remove the supernatant and repeat the washing 2x to minimize the debris in the pelleted cells. 
	NOTE: Always use fresh, warmed DPM for the cultured neurons. For the washing steps, DPM does not have to be fresh, but should be prewarmed to 37 &#176;C.</li>
	<li>Dilute the cells with fresh, warm DPM and transfer them to a microcentrifuge tube. The amount of DPM for dilution depends on the number of embryos used for tissue dissection. For example, use 150 &#956;L of DPM to dilute the cells obtained from ten embryos.</li>
	<li>Transfer 10 &#956;L of cells in DPM to a microcentrifuge tube. Mix them with 10 &#956;L of 0.4% Trypan blue stain. Count live (i.e, Trypan blue negative) cells using a hemocytometer or an automated cell counter. 
	NOTE: Use 30,000 cells for plating per well to obtain ~1,000 dopamine neurons per well. If the cell density is higher than ~30,000 cells per 6 &#956;L, further dilute the cells with DPM before plating so that the seeding volume is no less than 6 &#956;L.</li>
	<li>Without touching the bottom of the wells, remove the DPM from the micro islands created at step 1.6.</li>
	<li>In order to obtain reproducible cell density at each well, mix the cells by gentle pipetting prior to plating in the well. With a 1&#8211;10 &#956;L micropipette, add 6 &#956;L of cells to the middle of the well, at the location of each former micro island.</li>
	<li>Fill the empty wells at the edges of the plate with 150 &#956;L of water or 1x PBS to minimize evaporation from the wells containing neuronal cultures. Incubate the plate in an incubator at 37 &#176;C, 5% CO2 for 1 h.</li>
	<li>After 1 h, remove the plate from the incubator, add 100 &#956;L of DPM into each well with cells and place it back in the incubator.</li>
	<li>Two days after plating (day in vitro 2, or DIV2), remove 25 &#956;L and add 75 &#956;L of fresh DPM to bring the final media volume to 150 &#956;L and avoid evaporation as much as possible.</li>
	<li>Exchange half of the medium with fresh DPM (i.e., remove 75 &#956;L and add 75 &#956;L fresh DPM) at DIV5. Do not perform any media changes after DIV5.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4% Trypan Blue stain</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml CELLSTAR Polypropylene tube&#160;</td>
			<td>Greiner Bio-One GmbH, Frickenhausen, Germany</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M HCl</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>5 M NaOH</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>96-well ViewPlate (Black)</td>
			<td>PerkinElmer, Waltham, Massachusetts, USA</td>
			<td>6005182</td>
			<td></td>
		</tr>
		<tr>
			<td>99.5% Ethanol (EtOH)</td>
			<td>Altia Oyj, Rajam&#228;ki, Finland</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>AquaSil Siliconizing Fluid</td>
			<td>Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>TS-42799</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved 1.5 ml microcentrifuge tube</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>Bioruptor sonication device</td>
			<td>Diagenode, Liege, Belgium</td>
			<td>B01020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2+, Mg2+ free Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>14175-053</td>
			<td></td>
		</tr>
		<tr>
			<td>CellProfiler and CellAnalyst software packages</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>free open-source software</td>
		</tr>
		<tr>
			<td>Centrifuge 5702</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>5702000019</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>HeraCell, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting chamber</td>
			<td>BioRad Inc., Hercules, California, USA</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Ultra High Resolution Microscope with air table and cabinet</td>
			<td>GE Healthcare Life Sciences, Boston, Massachusetts, USA</td>
			<td>29254706</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease-I (Dnase I)</td>
			<td>Roche, Basel, Switzerland</td>
			<td>22098700</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>21331&#8211;020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco, Thermo Scientific, Waltham, Massachusetts, USA</td>
			<td>10500056</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospohate Buffer Saline (PBS)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Media kitchen, Institute of Biotechnology, University of Helsinki</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen, San Diego, California, USA</td>
			<td>ant-pm-1, ant-pm-2</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithine</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>11332481001</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin</td>
			<td>MP Biomedicals, Valiant Co., Yantai, Shandong, China</td>
			<td>2199700</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Er, S., et al. <a href="https://app.jove.com/v/61118">Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons.</a> J. Vis. Exp. (2020).
The video demonstrates the process of isolating and culturing dopamine neurons from the midbrain of mouse embryos. The isolated ventral midbrain floors are dissociated using enzymes. The resulting dopamine neurons are harvested and transferred into micro-islands inside multi-well plates. The cells are incubated with a dopamine neuron medium to maintain the cells at a high density inside the micro-islands.

<img alt="Figure 1" src="/files/ftp_upload/22633/22633_Figure1.jpg" /> 
Figure 1: Dissection of midbrain floor from E13.5 mouse embryo. (A) Cutting locations at the hindquarter of the head is marked with black arrows and white dashed lines. (B) The piece was removed from the rest of the embryo. The removed piece is circled. (C) The piece was turned 90&#176; to face the posterior towards the observer. (D</stro...

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

	Prepare dopamine ...

Take midbrain floor tissue isolated from the ventral region of the midbrain of mouse embryos.
The tissue is rich in developing dopamine neurons, which release the neurotransmitter dopamine.
Add an enzyme solution to break down the tissue matrix, thereby loosening the cells.
Add a serum solution containing DNase I and mechanically dissociate the digested tissue to generate a cell suspension. The serum proteins inhibit the enzymes, while DNase I degrades extracellular DNA released during cell lysis, preventing cell clumping.
Let the tissue debris settle and transfer the suspended cells to a fresh tube.
Centrifuge and remove the supernatant, then resuspend the cells in a dopamine neuron medium, or DPM.
Take a microplate coated with a cell culture substrate at the center of the wells, creating micro-islands.
Transfer the cells to the middle of the micro-islands to culture them at a high density.
Incubate, allowing the cells to adhere to the micro-islands.
Fill the wells with DPM and incubate, facilitating the growth of dopamine neurons.

41 조회수. 1차 배아 마우스 중뇌 도파민 뉴런의 분리 및 배양

비디오: 1차 배아 마우스 중뇌 도파민 뉴런의 분리 및 배양 - 실험

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16 스프라그 Dawley 쥐 배아에서 분리된 혼합 된 기본 해마 및 피질 뉴런에서 신경 구의 생성

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

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JoVE Journal

신경과학

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.

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 ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

Patch-Clamp 기록을 위해 신생아 설치류에서 전정 및 나선형 신경절 소마타를 분리 및 배양합니다.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons

내레이션 대본

Isolation and Culture of Primary Embryonic Mouse Midbrain Dopamine Neurons