spinal cord neuron isolation: a density gradient procedure to isolate neonatal spinal cord neurons for in vitro studies

Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies

To begin this procedure, separate the head from the body of the euthanized mouse pups using scissors. Next, stabilize the tail and arms on the procedure table with the dorsal side facing up. Then, cut the skin off using the curved iris scissors. After that, cut the spinal cord from the lumbar region just above the hips and then cut both sides of the thorax to separate it from the body.
Wash the samples sequentially in three 10-centimeter Petri dishes containing 5 milliliters of sterilized PBS for 10 seconds each to remove excess tissue. Subsequently, insert a 22-gauge needle and syringe filled with 5 milliliters of sterilized PBS into the caudal end of the spinal column and flush cranially, allowing the cord to exit into a fourth Petri dish.
Transfer the spinal cord in a 15-milliliter tube with 5 milliliters of HABG on ice. Avoid crushing the spinal cord and repeat the procedures for the rest of the pups. Ideally, this process should take no more than 30 minutes for one group of pups to ensure healthy neuron isolation.
To prepare the density gradient, prepare each of the four layers in four 15-milliliter tubes. Then, add 1 milliliter of each layer into a new 15-milliliter tube. Start with layer 1 at the bottom and add sequentially until reaching layer 4 at the top. Avoid disturbing the layers while adding and any vigorous movements that may cause mixing. Next, add the digestion media to the minced tissue. Place the sample in a shaker in the water bath at 30 degrees Celsius. Afterward, remove the tissue from the water bath and allow it to settle for a few minutes.
To perform trituration, aspirate the excess digestion medium. Suspend the tissue in 2 milliliters of HABG. Using a narrow-bore pipette, triturate 10 times in 45 seconds and avoid introducing air as it will significantly decrease the viable yield. Trituration is one of the most critical steps of the procedure. The tissue should be carefully drawn into and emptied from the fire-polished pipette, avoiding excess vigor, which can lead to neuronal death, or being too gentle, which will result in a low yield.
Following that, aspirate the top 2 milliliters of the supernatant and transfer it into a new 15-milliliter tube labeled &#34;collection&#34;. Repeat the collection procedures two more times to yield 6 milliliters of supernatant. Next, slowly transfer the collection tube contents into the gradient tube prepared previously, avoiding the disruption of the gradient.
To purify the neurons, centrifuge the gradient tube for 15 minutes at 800 x g and 22 degrees Celsius. Collect the desired layers and transfer them in a new 15-milliliter tube. For the highest purity neuron isolation, collect layer 3. For more yield with less purity, collect layers 2 to 3.
Next, dilute the density gradient by adding 5 milliliters of HABG to the newly collected layers. Centrifuge it at 200 x g for 2 minutes at 22 degrees Celsius. After 2 minutes, discard the supernatant and resuspend the cells in 5 milliliters of HABG by flicking the pellet. Subsequently, centrifuge the sample at 200 x g for 2 minutes at 22 degrees Celsius again. Then, discard the supernatant and resuspend the cells in 3 milliliters of neurobasal medium by flicking the pellet.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Harvesting Spinal Cords
NOTE: All instruments should be autoclaved (135 &#176;C and 30 psi for 4 x 7 min cycles) for sterility.
<ol>
	<li>Euthanize 1&#8211;3 day-old C57BL/6 mouse pups in a chamber with isoflurane. Wait 30 s after the cessation of movement and pinch the leg to confirm a lack of response.</li>
	<li>Separate the head from the body using scissors, with pup in the prone position.</li>
	<li>Stabilize the hind legs or tail and arms on the procedure table, with dorsal side facing the user.</li>
	<li>Cut the skin off using curved iris scissors.</li>
	<li>Cut the spinal cord from the lumbar region just above the hips and proceed to cut both sides of the thorax to separate it from the body. 
	NOTE: This requires the careful dissection of the spinal cord from visceral organs to avoid inadvertent damage to other organs (Figure 1a).</li>
	<li>Wash sequentially for 10 s in 3 x 10 cm Petri dishes containing 5 mL of 0.2 &#956;m filter-sterilized Phosphate-Buffered Saline (PBS) to remove excess tissue.</li>
	<li>Insert a 22G needle and syringe filled with 5 mL of filter-sterilized PBS into the caudal end of the spinal column and flush cranially, allowing the cord to exit into a fourth Petri dish (Figure 1b).</li>
	<li>Collect the spinal cord in a 15 mL tube with 5 mL of HABG (Table 1) on ice. Use care to avoid crushing the spinal cord.</li>
	<li>Repeat steps 1.1&#8211;1.8 for each pup in the litter. 
	NOTE: Ideally, this process should take less than 30 min per spinal cord to ensure healthy neuron isolation.</li>
</ol>
2. Isolating Neurons
NOTE: The following step should be performed in a laminar flow hood. Familiarity with basic sterile technique is expected.
<ol>
	<li>Tissue Mincing
	<ol>
		<li>Take the tube containing the spinal cords and shake lightly to suspend the tissue.</li>
		<li>Pour tissue from the tube into a 60 mm glass Petri dish and dice with a razor blade to create fine pieces ~0.5 mm in size.</li>
		<li>Transfer the tissue with a wide-bore pipette into a 15 mL tube containing 5 mL of HABG.</li>
		<li>Place it in a 30 &#176;C water bath for 30 min to allow the cells to equilibrate at this temperature. Keep the cells on a shaker just enough to allow them to be suspended in the fluid. 
		NOTE: This step is done to avoid shocking the cells upon transfer from ice to the digestion medium. Keeping the cells at 30 &#176;C helps to decrease cell death associated with an otherwise increased metabolism at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare the digestion medium (Table 1).</li>
	<li>Prepare the density gradient (Table 1).
	<ol>
		<li>Prepare each of the 4 layers in 4 separate 15 mL tubes, as outlined in&#160;Table 1.</li>
		<li>Add 1 mL from each layer into a new 15 mL tube. Start with layer 1 at the bottom and sequentially add until reaching layer 4 at the top. Avoid disturbing the layers while adding.</li>
	</ol>
	</li>
	<li>Wash the PDL-coated plates. Wash with sterile water for a few minutes before removing any remaining water and allowing them to dry for 1 h.</li>
	<li>Transfer the tissue to digestion medium.
	<ol>
		<li>Remove the tissue-containing tube from the shaker water bath at 30 &#176;C and allow it to settle for a few minutes.</li>
		<li>Remove the digestion medium tube from the 37 &#176;C water bath and aspirate it into a leur-lock syringe.</li>
		<li>Aspirate off the excess HABG from the tissue-containing tube.</li>
		<li>Use a leur-lock 0.2 &#956;m filter on the syringe to add digestion medium to the tissue-containing tube.</li>
		<li>Place the tube in a 30 &#176;C water bath for 30 min. Keep the cells shaking just enough to allow them to be suspended in the fluid. 
		NOTE: It is important not to keep the cells in the digestion medium for too long or to let the temperature get too high, which could lead to excessive digestion and result in the tissue becoming suspended in a gelatinous mixture.</li>
	</ol>
	</li>
	<li>During this period, coat the laminin.</li>
	<li>Perform trituration (i.e.&#160;separating the cells from the tissue).
	<ol>
		<li>Remove the tube from the shaking 30 &#176;C water bath and allow it to settle for a few min.</li>
		<li>Aspirate excess digestion medium.</li>
		<li>Suspend the tissue in 2 mL of HABG.</li>
		<li>Using a narrow-bore pipette, triturate 10x for 45 s. 
		NOTE: This is probably the single most crucial step and can significantly decrease the yield if not done properly.
		<ol>
			<li>Aspirate the tissue into the pipette and immediately empty the contents back.</li>
			<li>Avoid introducing air, as it will significantly decrease the viable yield. 
			NOTE: The ideal pipette is a 9&#34; glass pipette. The tip of the pipette should be fire polished to smooth out rough surfaces. It should then be siliconized by placement in a 1:20 solution of dichlorodimethylsilane (DMDCS) in chloroform and left O/N. The pipette should then be removed and allowed to air dry. Subsequently, it should be autoclaved for sterilization. 
			CAUTION: DMDCS and chloroform are highly flammable, and siliconizing should be carried out in a fume hood.</li>
		</ol>
		</li>
		<li>Aspirate the top 2 mL of supernatant and place it into a new 15 mL tube labeled &#34;collection&#34;.</li>
		<li>Repeat steps 2.7.3&#8211;2.7.5 two additional times (the cell collection tube should have 6 mL by the end).</li>
		<li>Slowly transfer the collection tube contents into the gradient tube prepared in step 2.2, avoiding the disruption of the gradient.</li>
	</ol>
	</li>
	<li>At this point, remove the previously prepared neurobasal medium (Table 1) from the refrigerator and allow it to warm in a 37 &#176;C bath.</li>
	<li>Purify the neurons.
	<ol>
		<li>Centrifuge the gradient tube for 15 min at 800 x g and 22 &#176;C.</li>
		<li>Collect the desired layer(s) with a pipette (Figure 2) and place in a new 15 mL tube. For the highest-purity neuron isolation (i.e.&#160;&#62;90%), collect layer 3. For more yield with less purity (i.e.&#160;&#62;70&#8211;80%), collect layers 2 &#38; 3.</li>
		<li>Dilute out the density gradient by adding 5 mL of HABG to the newly collected layers.</li>
		<li>Centrifuge at 200 x g for 2 min at 22 &#176;C.</li>
		<li>Discard the supernatant, resuspend in 5 mL of HABG, and flick the pellet to suspend the cells.</li>
		<li>Centrifuge at 200 x g for 2 min at 22 &#176;C.</li>
		<li>Discard the supernatant, resuspend in 3 mL of neurobasal medium, and flick the pellet to resuspend the cells.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media</td>
			<td>Storage</td>
			<td colspan="3">Ingredients</td>
			<td>Preparation</td>
		</tr>
		<tr>
			<td rowspan="4">HABG</td>
			<td rowspan="4">4 &#176;C/24h</td>
			<td></td>
			<td colspan="2">100 mL</td>
			<td rowspan="4">- Thaw B27 in 4 &#176;C fridge O/N 
			- Filter sterilize (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td>Hibernate A</td>
			<td colspan="2">97.8 mL</td>
		</tr>
		<tr>
			<td>B27 [2%]</td>
			<td colspan="2">2 mL</td>
		</tr>
		<tr>
			<td>GlutaMAX [0.5 mM]</td>
			<td colspan="2">0.25 mL</td>
		</tr>
		<tr>
			<td rowspan="5">Neurobasal Media</td>
			<td rowspan="5">4 &#176;C/1 week</td>
			<td></td>
			<td colspan="2">100 mL</td>
			<td rowspan="5">- Thaw B27 in 4 &#176;C fridge O/N 
			- Filter sterilize (0.22 &#181;m) 
			-&#160;Aliquot 50 mL portions into 50 mL tubes (store at 4 &#176;C)</td>
		</tr>
		<tr>
			<td>Neurobasal A</td>
			<td colspan="2">97 mL</td>
		</tr>
		<tr>
			<td>B27 [2%]</td>
			<td colspan="2">2 mL</td>
		</tr>
		<tr>
			<td>GlutaMAX [0.5 mM]</td>
			<td colspan="2">0.25 mL</td>
		</tr>
		<tr>
			<td>Pen/Strep [1%]</td>
			<td colspan="2">1 mL</td>
		</tr>
		<tr>
			<td rowspan="4">Digestion Media</td>
			<td rowspan="4">Day of Isolation</td>
			<td></td>
			<td colspan="2">10 mL</td>
			<td rowspan="4">- Shake vigorously 
			- Let it sit in 37&#176;C bath for 30 min (ideally prepared 30 min prior to use) 
			- Filter sterilize (0.22 &#181;m)</td>
		</tr>
		<tr>
			<td>HA-Ca</td>
			<td colspan="2">10 mL</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td colspan="2">25 mg</td>
		</tr>
		<tr>
			<td>GlutaMAX [0.5 mM]</td>
			<td colspan="2">0.025 mL</td>
		</tr>
		<tr>
			<td rowspan="7">Density Gradient</td>
			<td rowspan="7">Day of Isolation</td>
			<td></td>
			<td colspan="2">1 Gradient</td>
			<td rowspan="7"></td>
		</tr>
		<tr>
			<td rowspan="2">Layer</td>
			<td>OptiPrep</td>
			<td>HABG</td>
		</tr>
		<tr>
			<td>(0.13 mL)</td>
			<td>(5.29 mL)</td>
		</tr>
		<tr>
			<td>1 Bottom</td>
			<td>0.26 mL</td>
			<td>1.24 mL</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.19 mL</td>
			<td>1.31 mL</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.15 mL</td>
			<td>1.35 mL</td>
		</tr>
		<tr>
			<td>4 Top</td>
			<td>0.11 mL</td>
			<td>1.39 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Preparation of the media and solutions used in this protocol.

<table><tbody><tr><td>Hibernate A Medium - 500 mL</td><td>Thermo-Fisher</td><td>A1247501</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/A1247501&lt;/a&gt;</td></tr><tr><td>Hibernate A Minus Calcium - 500 mL</td><td>Brainbits</td><td>HA-Ca</td><td>&lt;a href=&quot;http://www.brainbitsllc.com/&quot;&gt;http://www.brainbitsllc.com/ hibernate-a-minus-calcium/&lt;/a&gt;</td></tr><tr><td>Glutamax 100X - 100 mL</td><td>Thermo-Fisher</td><td>35050061</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/35050079&lt;/a&gt;</td></tr><tr><td>B27 Supplement 50X - 10 mL</td><td>Thermo-Fisher</td><td>17504044</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/17504044&lt;/a&gt;</td></tr><tr><td>Papain, Lyophilized - 100 mg</td><td>Worthington</td><td>LS003119</td><td>https://www.worthington-biochem.com/PAP/cat.html</td></tr><tr><td>Neurobasal A Medium - 500 mL</td><td>Thermo-Fisher</td><td>10888022</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/10888022&lt;/a&gt;</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ mL)</td><td>Thermo-Fisher</td><td>15140122</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/15140122&lt;/a&gt;</td></tr><tr><td>Poly-D-Lysine (PDL) hydrobromide- 5 mg</td><td>Sigma-Aldrich</td><td>P6407-5MG</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/p6407? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Mouse Laminin - 1 mg</td><td>Thermo-Fisher</td><td>23017015</td><td>&lt;a href=&quot;http://www.thermofisher.com/&quot;&gt;https://www.thermofisher.com/ order/catalog/product/23017015&lt;/a&gt;</td></tr><tr><td>Trypan Blue - 20 mL</td><td>Sigma-Aldrich</td><td>T8154-20ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/t8154? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>OptiPrep Density Gradient Medium-250 mL</td><td>Sigma-Aldrich</td><td>D1556-250ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/d1556? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Dichlorodimethylsilane (DMDCS, Sigma Silicoat)</td><td>Sigma-Aldrich</td><td>440272-100ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/aldrich/440272? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>288306-1L</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sial/288306? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Glass Pippette - 9&quot;</td><td>Sigma-Aldrich</td><td>13-678-20C</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/cls7095d9? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Pipette bulb - 5 mL</td><td>Sigma-Aldrich</td><td>Z186678-3EA</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/aldrich/z186678? lang=en&amp;amp;region=US&amp;amp;cm_sp=Insite-&lt;br/&gt; _-prodRecCold_xviews-_- prodRecCold10-1&lt;/a&gt;</td></tr><tr><td>BRAND&lt;sup&gt;&amp;reg;&lt;/sup&gt; Petri dish, glass - 60 x 15 mm</td><td>Sigma-Aldrich</td><td>BR455717-10EA</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/aldrich/br455717? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr><tr><td>Sterile 24-Well Cell Culture Plate</td><td>Sigma-Aldrich</td><td>M8812-100EA</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com/ catalog/product/sigma/m8812? lang=en&amp;amp;region=US&lt;/a&gt;</td></tr></tbody></table>

Source: Eldeiry, M. et al. <a href="https://www.jove.com/video/55856" target="_blank">Spinal Cord Neurons Isolation and Culture from Neonatal Mice.</a> J. Vis. Exp. (2017)
In this video we describe a step-by-step protocol to isolate spinal cord neurons from a neonatal mouse for in vitro studies.

<img alt="Figure 1" src="/files/ftp_upload/20698/20698_Figure1.jpg" /> 
Figure 1: Spinal column dissected and spinal cord released from a 3 day-old neonatal mouse. The arrow in (a) points towards the caudal end of the spine, where a needle is inserted to release the spinal cord. A released spinal cord (b) is shown submerged in PBS.
<img alt="Figure 1" src="/files/ftp_upload/20698/20698_Figure2.jpg" /> 
Figure 2: Density gradient, with cells added-on after centrifugation. The layers are outlined and better visualized on the cartoon depiction. The most superficial layer (0) is generally debris from the spinal cord tissue. Layer 1 is rich in oligodendrocytes and astrocytes. Layers 2 and 3 contain the majority of neurons. While layer 3 contains neurons with the highest purity, some supporting cells (i.e., astrocytes and oligodendrocytes) are found in layer 2. The pellet at the bottom mainly contains microglial cells.

[INSERT FIGURE1 HERE]

Figure 1: Spinal column dissected and spinal cord released from a 3 day-old neonatal mouse. The arrow in (a) points towards the caudal end of the spine, where a needle is inserted to release the spinal cord. A released spinal cord (b) is shown submerged in PBS.

[INSERT FIGURE2 HERE]

Figure 2: Density gradient, with cells added-on after centrifugation. The layers are outlined and better visualized on the cartoon depiction. The most superficial layer (0) is generally debris from the spinal cord tissue. Layer 1 is rich in oligodendrocytes and astrocytes. Layers 2 and 3 contain the majority of neurons. While layer 3 contains neurons with the highest purity, some supporting cells (i.e., astrocytes and oligodendrocytes) are found in layer 2. The pellet at the bottom mainly contains microglial cells.

Source: Eldeiry, M. et al. Spinal Cord Neurons Isolation and Culture from Neonatal Mice. J. Vis. Exp. (2017)
In this video we describe a step-by-step ...

To isolate spinal cord neurons, take a vertebral column of a neonatal mouse and place it onto a Petri dish. Flush the vertebral column with saline buffer to release the spinal cord. Transfer the spinal cord into cold neuronal medium to promote cell survival.
Next, mince the spinal cord and transfer the fragments into a tube containing fresh neuronal&#160; medium. Incubate the tube in a shaker water bath for acclimatization. After incubation, remove the spent medium and add the digestion medium. Digestive enzymes cleave the tissue matrix to loosen up the cells.
Perform trituration by aspirating the tissue fragments into the pipette and swiftly empty the components, releasing individual cells into suspension. Then, transfer this suspension into a tube containing density gradient medium and centrifuge.
Following centrifugation, the tube&#39;s superficial layer contains tissue debris. The first layer includes oligodendrocytes and astrocytes. The second and third layers comprise neurons, with the third layer having the highest purity. Lastly, the pellet contains microglial cells.
Collect the third layer into a fresh tube and add neuronal medium to dilute out the density gradient medium. Centrifuge to pelletize the neurons. Discard the supernatant and resuspend the pellet in a neurobasal medium to preserve the neurons for further experiments.

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Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies

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Spinal Cord Neuron Isolation: A Density Gradient Procedure to Isolate Neonatal Spinal Cord Neurons for In Vitro Studies