Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined.
The overall goal of this experiment is to label and isolate the airway-innervating nodose neurons of the vagus nerve. This method can help answer key questions in the Peripheral Neurobiology field. Such as, what identifying properties do airway-innervating neurons possess?
The main advantage of this technique is that it provides a robust and high throughput method for targeting and isolating specific neuronal populations. After harvesting the sensory ganglia, aspirate 500 microliters of ganglia dissociation solution from each tube, without disturbing the ganglia. Next, add one 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 one milliliter of ganglia dissociation solution supplemented with digestion enzyme to the neurons, followed by the addition of 20 microliters of Fast Blue to the positive control tube. 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. Next, label 1.5 milliliter collection tubes for sorting, including one Fast Blue positive and one Fast Blue negative tube per sorting sample.
At the end of the digestion period, discard the digestion enzyme and wash the ganglia two times with one 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. Filer 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. While the cells are spinning add 300 microliters of freshly prepared lysis buffer to the Fast Blue positive collection tube and 600 microliters of lysis buffer to the negative collection tube to ensure that the sorting sheath fluid does not significantly dilute the lysis buffer.
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.
Then re-suspend the cells in 200 to 300 microliters of sorting ganglia dissociation solution with a 1, 000 microliter pipette set to 200 microliters. And place the cells on ice until they are ready for sorting. After purifying the ganglia cells by FACS, homogenize the neurons in lysis buffer for one minute with vortexing.
Next, mix the cells several times with one volume of 70%ethanol and transfer up to 700 microliters of the sample to a spin column. Centrifuge the column for 15 seconds at 8, 000 x g in a microcentrifuge and discard the flow through. When the entire sample has been passed through the spin column, extract the RNA according to standard RNA purification protocols.
Then use a microfluidic electrophoresis system to test the RNA quality according to the manufacturer's specifications. In these graphs, the negative and positive Fast Blue controls used to set the sorting gates are shown. The dissociated Fast Blue Labeled Nodose cells can then be sorted into Fast Blue positive and Fast Blue negative populations.
The sorting efficiency for this method is 73 to 85%with approximately 1, 500 or fewer Fast Blue positive and nearly 60, 000 Fast Blue negative cells collected on average. As observed for this representative sample, the 18S and 28S peaks attained by microfluidic electrophoresis can then be used to calculate the RNA integrity number to determine the quality of the extracted RNA. Once mastered, this technique can be completed in four and a half hours if it's performed properly.
While attempting this procedure, it's important to remember to move through the steps quickly and to use freshly prepared solutions for RNA extraction. Following this procedure, other methods like neuronal culturing can be performed to answer additional questions, like what functional properties are unique to airway-innervating neurons? After its development, this technique paved the way for researchers in the field of Peripheral Neurobiology to explore the unique properties of a specific airway-innervating population of neurons in the nodose ganglia of the vagus nerve.
After watching this video, you should have a good understanding of how to label and dissociate specific neurons of the nodose ganglia which can then be used for RNA sequencing, or plated for functional studies. Don't forget that working with 2-Mercaptoethanol can be extremely hazardous, and that precautions such as wearing gloves and working in a chemical hood should always be taken when using this compound.
