The overall goal of this procedure is to obtain enough cells from dorsal root ganglions and sciatic nerves from an individual mouse so as to be able to perform flow cytometry analysis of the cells. Generally, individuals new to this method will struggle because peripheral neuronal material generates large amounts of debris when digested. The main advantage with this method is that several markers can be used simultaneously to identify multiple cell types.
The implications of this technique extend towards the diagnosis of inflammation and immunopathology during neuropathy. Whilst this method can provide insight to murine models, it can also be applied to other organisms, such as human and pig nerves, with minor modifications. Generally, individuals new to the method, will struggle due to the large amounts of debris generated by the digestions.
Visual demonstration of this method is critical as the dissection and cell dissociation steps are difficult to perform because the neuronal material per mouse is limiting. We first had the idea for this method when we saw a protocol isolating swan cell from sciatic nerve from Steve Lacroix's lab. To begin, prepare the mouse as described in the accompanying text protocol and position it ventral side down while stretching out the lower limbs.
Next, use a pair of forceps to bilaterally dissect the whole sciatic nerves. Start by separating the muscle tissue from the upper dorsal thigh and follow the nerve up along the spinal column. Cut the nerve where it exits from the spinal column and immediately above the knee at the other end.
Then transfer the excised nerves to a 1.5 milliliter tube containing 1 milliliter of ice cold PBS. Store the nerves on ice. Next, isolate the dorsal root ganglion.
Start by cutting the ribs parallel with and close to the spinal column on both sides. Then, transversely cut the spinal column at the level of the femurs on the posterior side and close to the skull on the anterior side. Remove the spinal column and use iris scissors to cut away all muscle, fat, and other soft tissues from the spine.
Next, place the spinal column with the ventral side facing up and open it starting with caudal end by sliding one tip of the scissors into the vertebral canal at the 12 o'clock position and snipping the bone. Proceeding until the whole spine is cut, repeat this procedure at the six o'clock position until the spine is halved. Separate the two halves of the spine, then place the sample under a dissection microscope.
Remove the spinal cord to expose the spinal nerves, then locate and remove the lumbar dorsal root ganglions by gently tugging on the dorsal root to pull it into the vertebral canals. Next, use iris scissors to clip the nerves and connective tissue around the dorsal root ganglion. Using forceps, transfer the dorsal root ganglions to a 1.5 milliliter tube and store them on ice.
To locate the dorsal root ganglions, extract them without damaging them, and free them from the spinal nerve, requires exercise and skill. Prepare a digestion media, supplement it with one hundred micrograms per milliliter of deoxyribonuclease and store it on ice. Then, add 500 microliters of digestion media into each of the tubes with the sciatic nerves and the dorsal root ganglions.
For the sciatic nerves, chop the material 15 to 20 times using a pair of spring scissors, and then incubate the tissue suspensions in a shaking incubator at 37 degrees Celsius for 20 minutes with gentle agitation. Following incubation, repeatedly triturate the cells suspension through a 1, 000 microliter pipette tip to mechanically dissociate any incompletely digested tissue. Repeat the trituration through a 1, 000 microliter pipette tip.
Then incubate the sample for an additional 10 minutes at 37 degrees celsius with gentle agitation. Prepare the ice cold FACS buffer and use it to rinse and soak a stainless steel screen with a mesh size of 140 microns in a 60 millimeter petri dish on ice. Then, use blunt forceps to hold the 140 micron screen filter over the petri dish and repeatedly pass the tissue suspensions through the filter.
Collect the cell suspension from the petri dish and transfer it to the same 1.5 milliliter tube stored on ice. Push any incompletely digested tissue that remains on the filter through by repeated trituration with a 1, 000 microliter pipette tip. Then, rinse the screen filter twice with 500 microliters of FACS buffer and combine it with the cell suspension in the 1.5 milliliter tube.
If too much of the sample is incompletely digested and passed through the metal mesh, cell loss may occur and flow cytometry analysis may be impossible. Centrifuge the cell suspensions and discard the supernatant. Then re-suspend the cell pellet in one milliliter of FACS buffer.
Repeat this washing step once, re-suspending the final cell pellet in 150 to 350 microliters of FACS buffer. The cells are now ready for staining. At the computer, select the appropriate channels for detection of the fluorophores of interest, the appropriate flow rate of the samples, and the total number of events to be collected per sample.
The minimum number of events should be one million. Next, create scatter plots under a global worksheet of the side scatter A versus the Forward Scatter A as well as for each fluorophore of interest versus the Forward Scatter A.Also, create a statistical view that will display the number of events and the mean fluorescent intensity for the selected fluorophores. Determine the parent gate, P1, based on scatter plot from DAPI only staining controls.
Attach the sample to flow cytometry and click on acquire. Observe the DAPI versus FSC plot and reduce the voltage of the DNA pacific blue channel until the DAPI+population becomes visible. Using the rectangle gating tool, draw a box around the DAPI+population.
This represents the P1 gate. Select the scatter plots for each of the fluorophores and display only the DAPI+events. This represents the background fluorescence for each of the fluorophores.
Using the rectangle gating too, draw a box in the scatter plots for each of the fluorophores, starting at 103 on the y-axis. This gate represents the region in which positive events for each of the stated markers should be found. Adjust the voltages for each of the fluorophores using the DAPI only staining controls.
Attach the sample to flow cytometry and click on acquire. Observe the position of the DAPI+population for each of the fluorophores and adjust the respective voltages such that the populations fall outside of the rectangle gate. Once the flow cytometer has been set up, run the samples and export the Forward Scatter files for further analysis.
Washing out the DAPI stain prior to analysis by flow cytometry decreased the fluorescent intensity of the nonnucleated debris. This allowed the material on the high end of the Forward Scatter axis, which contains incompletely digested tissue, to be excluded in the selection of the singlet population. Overall, more than 30, 000 singlets can be regularly obtained from two full sciatic nerves and more than 200, 000 singlets can be obtained from the dorsal root ganglions.
From the sciatic nerves, approximately 5%of the singlets expressed CD45 on their surface, whereas approximately 5%to 10%were found to express it in the dorsal root ganglions thereby defining these cells as leukocytes of hematopoietic origin. A majority of the CD45 positive singlets also expressed MHC class II in a gradual manner. In the dorsal root ganglions, CD11b can be used to detect microglial like cells, however;this microglia population is absent from the sciatic nerves.
In the sciatic nerve and dorsal root ganglion, 2%to 5%of the singlets expressed CD68. In the sciatic nerves approximately 60%80%of the CD68 positive macrophages expressed CD206. While a smaller proportion of the CD68 positive co-expressed CD206 in the dorsal root ganglion.
While performing this procedure, it's important to keep a consistent temperature in order to ensure a consistent result. After it's development this technique allowed for researchers in the field of diabetes to explore the toxic effects of streptozotocin used to induce diabetes in mice. After watching this video, you should have a good understanding of how to perform flow cytometry on peripheral neuronal tissue.
Don't forget that working with paraformaldehyde can be hazardous and precautions should always be taken, such as wearing gloves and safety goggles.
