To investigate molecular and cellular mechanisms in autoimmune diseases such as multiple sclerosis, the generation of reproducible and sophisticated cell isolation protocols is an unmet need. Most of the current technologies to isolate and analyze CNS-resident cells show serious shortcomings, such as focusing on only one cell type or only postnatal mice. The current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to analyze complex neuronal networks and new inflammatory pathways ex vivo from one CNS cell's suspension applicable in healthy mice and those with experimental autoimmune encephalomyelitis.
Additionally, mice numbers are decreased. Our protocol for the simultaneous isolation of all four major CNS-resident cell types in healthy and EAE mice could be used as a helpful tool for research groups studying new inflammatory pathways, allowing a much more accurate analysis of complex biomolecular mechanisms and cellular networks ex vivo. New scientific questions that could be answered with the help of our protocol are dynamic investigations in EAE during different stages of disease course like neuroinflammation, neurodegeneration, and remission.
Also cell-cell interactions and biochemical pathways could be studied on an individual level. Likewise, functional assays could be performed with cultivated cells. We intend to focus on dynamic studies of the EAE course for multiomic analysis from one individual CNS homogenate per EAE time point.
Begin by placing the mouse in supine position and fixing the limbs with needles. Apply 75%ethanol on the front body of the animal. Make a longitudinal section through the skin and fascia on the abdomen and thorax.
Cut the ribs parasternal and fold up the thorax to gain free access to the heart. Fix the thorax folded upwards with needles. Open the right atrium using scissors.
Using a cannula, apply 20 milliliters of one time PBS into the left ventricle to flush out the blood through the incised right atrium. Make a longitudinal section by cutting the skin on top of the head of the mouse to expose the skull and shift the skin around the head using forceps. Incise the skull with the help of a scissor along the sagittal suture.
Insert the tip of the scissors along the incision line to crack open the calotte. Remove the remaining parts of the calotte with forceps so that the brain is fully exposed. Remove the brain carefully and place it into a murine brain matrix.
Cut the brain into one-millimeter-thick sagittal slices by using a razor blade. With the help of scissors, cut the vertebral column just above the iliac crest. Insert a 20-gauge needle attached to a 20-milliliter syringe containing one time PBS into the spinal canal.
Flush the spinal cord out of the spinal canal from caudal to cranial. Cut the spinal cord into 0.5-centimeter-long segments using a scalpel. Store the CNS cell suspension consisting of the brain and corresponding spinal cord in a Petri dish containing three milliliters of cold DPBS.
To begin, dissect the brain and spinal cord tissue and store the cell suspension on ice. Then prepare an appropriate volume of enzyme mix one and two. Transfer 1950 microliters of enzyme mix one into the C tube and add the tissue pieces of the CNS cell suspension.
Then add 30 microliters of enzyme mix two to the C tube. Close the C tubes tightly and attach them upside down onto the sleeve of the cell dissociator with heaters. Run the appropriate program and observe for at least five minutes to ensure all tubes turn at the same velocity.
In the meantime, place a 70-micron strainer on a 50-milliliter tube and pre-moisten the strainer with two milliliters of DPBS. After termination of the program, detach the C tubes from the dissociation and centrifuge the samples at 300 g for one minute at four degrees Celsius. Re-suspend the sample and apply it to the pre-moistened strainer.
Add 10 milliliters of cold DPBS to the empty C tube, mix, and add the suspension onto the corresponding strainer. After discarding the strainers, centrifuge the cell suspension again for 10 minutes and then carefully aspirate the supernatant to obtain a cell pellet. To begin, obtain a cell pellet after dissociation of CNS tissue and re-suspend it with 3100 microliters of DPBS in one 15-milliliter tube, do not vortex.
Add 1800 microliters of the debris removal solution from the adult brain dissociation kit to two pooled CNS cell suspensions. Invert the tube and mix the suspension, then gently overlay it with four milliliters of cold DPBS and observe a clear gradient. Centrifuge the tubes for 10 minutes at 3000 g at four degrees Celsius with full acceleration and no break.
After centrifugation, three phases are formed. Aspirate the two top phases completely and discard them, ensuring no myelin residues are left behind. Fill up the tube with cold DPBS up to 14 milliliters and close it.
Invert the tube with force on the workbench until the cell pellet becomes detached from the bottom of the tube. Centrifuge the sample and aspirate the supernatant completely. For red blood cell removal, add one milliliter of the red blood cell removal solution to the cell pellet.
After re-suspending the pellet, incubate the solution for 10 minutes at four degrees Celsius. Then add 10 milliliters of cold PB buffer, centrifuge the sample, and completely aspirate the supernatant. For cell counting, dilute the cell suspension by 50 times in PB buffer and then by 1 to 10 dilution in 0.4%Trypan Blue solution.
Then determine the cell count using an improved counting chamber. To begin, divide the purified undiluted mouse CNS cell suspension into two fractions for further isolations of microglia and oligodendrocytes. Then add magnetically labeled microbeads specific for the surface antigen for different CNS cell types to the respective cell suspension.
Place the column in the magnetic field, equilibrate the column, and then add the resulting cell suspension onto the column. Magnetically labeled cells will be retained within the column and unlabeled cells will run through. After removing the column from the magnetic field, flush out magnetically labeled cells from the column into a tube as the positively selected cell fraction.
Divide the negative flow through from the oligodendrocytes into two fractions for the further isolations of neurons and astrocytes. Flow cytometry analysis using cell type specific markers combined with live or dead cell discrimination yielded microglia, oligodendrocytes, astrocytes, and neurons. Phenotypic characterization of the different cell populations prove that single-cell suspensions with approximately 90%purity and viability were obtained for all main CNS-resident cell types.
