isolation of cells from sensory ganglia

Isolation of Cells from Sensory Ganglia

After harvesting the sensory ganglia, aspirate 500 microliters of ganglia dissociation solution from each tube without disturbing the ganglia. Next, add 1 milliliter of PBS to each sample and wait for the ganglia to settle to the bottom of the tubes.
Then, remove the PBS and add 1 milliliter of ganglia dissociation solution supplemented with digestion enzyme to the neurons. Incubate the tubes at 37 degrees Celsius for the appropriate time, based on the age of the digestion enzyme, shaking and flicking the tubes briefly every 15 minutes to ensure the ganglia are covered by the digestion solution.
While the nerve cells are being digested, slowly and carefully add 400 microliters of freshly prepared 12% density solution over 400 microliters of 28% density solution in one 1.5-milliliter tube per sample. Two distinct layers should be visible, one darker than the other.
At the end of the digestion period, discard the digestion enzyme, and wash the ganglia two times with 1 milliliter of PBS as just demonstrated. After the second wash, add 200 microliters of fresh ganglia dissociation solution to each tube.
Then, using a 200-microliter pipette set to a 100-microliter volume, pipette the ganglia up and down several times to dissociate the cells into a single-cell suspension, taking care to avoid bubbles. When no intact tissue pieces are visible, holding a 70-micron cell strainer firmly in one hand, pipette the dissociated cell solution into the center of the strainer. Then, wash the dissociation tube with 100 microliters of fresh ganglia dissociation solution to collect any additional cells.
Filter the wash through the strainer, and use a new pipette tip to aspirate any remaining cell-containing solution on the underside of the strainer, adding it to the cell suspension. Now, carefully layer all 300 microliters of the cells on top of the previously prepared density gradient, and collect the ganglia cells by centrifugation.
At the end of the centrifugation, carefully remove the top 700 microliters of solution containing the majority of the cell debris. Then, mix 700 microliters of fresh ganglia dissociation solution with the remaining cells. Pellet the cells by centrifugation, and carefully discard the supernatant.

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All procedures involving sample collection have been performed in accordance with the institute&#8217;s IRB guidelines.
1. Sensory Ganglia Dissociation
<ol>
	<li>Pipette out 500 &#181;l Ganglia Dissociation Solution (GaDS) (Table 1), leaving ganglia in the tube. Wash ganglia once by adding 1 ml Phosphate buffer solution (PBS), wait for ganglia to settle to the bottom of the tube, and then pipette out PBS.</li>
	<li>Add 1 ml GaDS and 22 &#181;l of digestion enzyme (collagenase I/II and protease mix, 2.5 mg/ml in water) to each tube.</li>
	<li>Add 20 &#181;l Fast Blue (5.26 mM) to the positive control tube.</li>
	<li>Put the tubes in a water bath or heating block set at 37 &#176;C.</li>
	<li>Every 15 min, shake the tubes briefly and flick them to ensure the ganglia are covered by the solution. 
	Note: The time of ganglia digestion depends on the age of the digestion enzyme. Within one month of dissolving the digestion enzyme, digest for 30 min; within 2 - 6 months, digest for 45 min. If the enzyme is over six months old, digest the ganglia for 60 min.</li>
	<li>While ganglia are being digested, prepare density gradients using a particle solution (colloidal silica particles coated with polyvinylpyrrolidone).</li>
	<li>Mix 12% particle solution in GaDS, 500 &#181;l per sample.</li>
	<li>Mix 28% particle solution in GaDS, 500 &#181;l per sample.</li>
	<li>Slowly and carefully add 400 &#181;l 12% density solution on top of 400 &#181;l 28% density solution in a fresh 1.5 ml tube for each sample. Do not allow the two layers to mix. Two distinct layers should be visible in the tube, one darker than the other.</li>
	<li>After the appropriate digestion time, remove GaDS with digestion enzyme and wash ganglia twice with 1 ml PBS. Add 200 &#181;l of fresh GaDS to each tube.</li>
	<li>Use a 200 &#181;l pipette, set to 100 &#181;l volume, and pipette ganglia up and down several times to break cells apart. Repeat until no intact piece of tissue is seen. Avoid creating bubbles in the solution.</li>
	<li>Pass dissociated cells through a 70 &#181;m cell strainer.</li>
	<li>Add another 100 &#181;l GaDS to the original dissociation tube to pick up any remaining stray cells left and pass the additional solution through the cell strainer. Using a new pipette tip, collect any remaining cell-containing liquid that has passed through the strainer and clung to the mesh. The final volume of cells suspended in GaDS is 300 &#181;l.</li>
	<li>Carefully layer the 300 &#181;l of cells on top of the previously made density gradient. Avoid bubbles and mixing of cells with the density layers.</li>
	<li>Centrifuge for 10 min at 2,900 x g at RT.</li>
	<li>Once centrifugation is complete, carefully remove and discard the top 700 &#181;l layer. This layer contains the majority of cell debris that will otherwise interfere with cell sorting.</li>
	<li>Add 700 &#181;l fresh GaDS to the remaining cell containing solution and pipette up and down several times to mix.</li>
	<li>18. Centrifuge for 15 min at 2,900 x g to pellet cells.</li>
	<li>19. Once centrifugation is done, carefully remove and discard the supernatant, leaving the cell pellet.</li>
	<li>Resuspend the cell pellet in 200 - 300 &#181;l GaDS by pipetting up and down with a 1,000 &#181;l pipette set to 200 &#181;l several times. Put samples on ice.</li>
</ol>
Table 1. Reagent Mixture for Ganglia Dissociation Solution (GaDS).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ingredient</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>Advanced DMEM/f12</td>
			<td>9.5 ml</td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>HEPES (10 mM)</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>100 &#181;l</td>
		</tr>
		<tr>
			<td>B27 (no vitamin A)</td>
			<td>200 &#181;l</td>
		</tr>
		<tr>
			<td>NGF (50 &#181;g/ml)</td>
			<td>10 &#181;l</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS) Ca and Mg free</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamine (Glutamax)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 (no vitamin A)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Nerve Growth Factor (NGF)</td>
			<td>Sigma</td>
			<td>N6009</td>
			<td>stock 50 &#181;g/ml in PBS/10% FBS</td>
		</tr>
		<tr>
			<td>digestion enzyme, Liberase DH Research Grade</td>
			<td>Roche</td>
			<td>5401054001</td>
			<td>stock 2.5 mg/ml in water</td>
		</tr>
		<tr>
			<td>particle solution (Percoll)</td>
			<td>Sigma</td>
			<td>P1644-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>LabNet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70 um cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kaelberer, M. M., et al. <a href="https://app.jove.com/v/53917">A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.</a> J. Vis. Exp. (2016).
This video demonstrates the isolation of cells from the sensory ganglia of mice. Ganglia are subjected to enzymatic digestion to dissolve the extracellular matrix. Single cells are then collected via filtering through a cell strainer followed by density gradient centrifugation for debris removal. Finally, washing and centrifugation are done to collect the isolated cells for future analysis.

All procedures involving sample collection have been performed in accordance with the institute&#8217;s IRB guidelines.
1. Sensory Ganglia Dissociation

	...

Take prewashed sensory ganglia in a tube.
Add a dissociation solution containing a mixture of digestive enzymes and incubate.
These enzymes break down the extracellular matrix, releasing individual cells.
After incubation, remove the dissociation solution.
Wash the ganglia with a phosphate buffer, and add a fresh dissociation solution.
Pipette the ganglia up and down to dissociate the cells.
Pass the suspension through a cell strainer to remove any clumps.
Collect any remaining cells from the bottom of the strainer and put them into the tube.
Layer the cell suspension over a density gradient tube and centrifuge.
Based on the differences in shape and mass, cells migrate to the lower layer, while cellular debris remains in the upper layer.
Discard the upper layer.
Add fresh medium to the remaining cell suspension and centrifuge.
Discard the supernatant and store the sensory ganglia cells for further use.

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JoVE Journal

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The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

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Isolation of Cells from Sensory Ganglia

TRANSKRIPT

Isolation of Cells from Sensory Ganglia