The overall goal of this experiment is to assess the various markers expressed by macrophage subpopulations within the central nervous system or CNS. This method can help answer key questions in the neuroimmunology field, such as duperry macrophages as we try to CNS doing dizzies, and what are the phenotypes of CNS in macrophages. The main advantages of this technique are the isolation of a large number of highly purified macrophage sub-population, as well as a contification of macrophage sub-population cell marker.

Begin by using fine scissors to cut the ventral skin just below the xiphoid process to expose the thoracic cavity. Make an incision in the diaphragm, and cut the lateral aspects of the ribcage in a caudal to rostral direction to expose the heart. After identifying the left ventricle and the right atrium, insert a butterfly catheter with a 25 gauage needle into the left ventricle.

Make an incision on the right atrium and begin profusing 20 milliliters of PBS through the catheter at a rate of ten milliliters per minute as soon as the blood begins to flow. A successful profusion will be indicated by a blanching of the lungs and the liver as the blood is displaced. Next, use fine scissors to dissect the spinal column.

Excising the abdominal wall musculature on the ventral side of the spinal column, and cutting the vertebral column at the base of the tail to remove the spine as it is harvested. Insert a ten milliliter syringe of PBS equipped with a modified 200 microliter pipette tip into the lumbar side of the spinal column. Then flush the spinal cord on the cervical side.

Remove the skin above the skull, and insert one tip of the scissors at the base of the skull. Cut the cranium following the sagittal suture. Then insert small forceps into the cut and gently remove the top of the skull.

Dissect the brain from the mouse head and remove the olfactory bulb and cerebellum. Then place the brain and spinal cord in a one point five milliliter tube containing one point one two eight milliliters of digestion cocktail. To dissociate the cells, use small scissors to finely cut the brain and spinal cord into one to two cubic millimeter pieces and place the pieces at 37 degrees Celsius and five percent Carbon Dioxide for 30 minutes.

At the end of the incubation, stop the digestion with 20 microliters of zero point five molar EDTA, followed by gentle trituration with a five milliliter pipette. Filter the resulting cell suspension through a 70 micron mesh strainer into a 50 milliliter conical tube using the plunger of a ten milliliter syringe to gently macerates any remaining brain or spinal cord tissue. Rinse the strainer with 20 milliliters of five percent FBS and PBS.

And collect the filtered cells by centrifugation. Then, carefully aspirate the supernatant without disturbing the pellet. For density gradient separation of the cells, we suspend the pellet with four milliliters of 30 percent density gradient medium, and transferred the cells into a 15 milliliter conical tube.

Gently underlay four milliliters of 37 percent density gradient medium, followed by four milliliters of 70 percent density gradient medium under the 37 percent density gradient medium. Transfer the tube to a bench-top centrifuge to separate the cells. The microglia and macrophage cells will be at the interface of the 70 and 37 percent density gradient layers.

The tube will appear stratified. After carefully removing the upper layer of myelin, transfer three to four milliliters of the microglia macrophage interphase layer into a new 15 milliliter tube. Then wash the immune cells with three centrifuges in 15 milliliters of PBS, re-suspending the pellet in one milliliter of PBS after the final wash.

To asses the macrophage sub-population marker expression, transfer the cells into a five milliliter flow cytometry analysis tube, followed by the addition of one microliter of fixable dead cell stain reagent. After 30 minutes on ice, protected from light, wash the samples in two milliliters of PBS, and re-suspend the pellet in 400 microliters of fax buffer per experimental analysis tube. Split the cells into the appropriate number of five milliliter flow cytometry analysis tubes.

Then block the non-specific FC receptor binding with one microgram of CD 16 CD 32 blocking antibody cocktail per 100 microliters of cells for a 10 to 15 minute incubation on ice, protected from light. Next, label the cells with 100 microliters of the appropriate primary antibodies, for a 30 minute incubation on ice, protected from light. At the end of the incubation, wash the cells in two milliliters of PBS four times.

Then add 100 microliters of the appropriate secondary antibodies for 30 minutes on ice, protected from light, followed by four washes in PBS as just demonstrated. After the last wash, gently re-suspend the pellets in 100 microliters of one percent paraformaldehyde for 15 minutes at room temperature, protected from light. Then wash the cells four times in PBS.

Re-suspend the final pellets in 200 microliters of fresh PBS, and analyze the cells by flow cytometry. Brain-and spinal cord-derived microglia and monocyte-derived macrophages are identified based on their CD 11 B and CD 45 expression. The expression of CX3CR1, and Tmem119, by the CD 11 B positive CD 45 medium microglial cells allows this cell population to be further distinguished from the CD 11 C positives CD 45 high monocyte-derived macrophages, which do not express either of these markers, but do express CCR2.

After density gradient separation, 85 to 95 percent of the cells are still viable. In the absence of density gradient isolation, the macrophage subpopulations are not identifiable in a forward bi-side scatter plot, because of the presence of non-macrophage cells, and myelin debris. As the epitope recognized by the anti-Tmem119 antibody is intracellular, permeabilization is required for labeling of this important microglial cell marker.

Note that permeabilization induces cell shrinkage, however, causing the macrophage subpopulations to appear smaller by forward versus side scatter analysis. Once mastered, this technique can be completed in five hours if it is performed properly. While I attempted this procedure, it's important to remember to set up the density gradiance very carefully.

Following this procedure, other methods, like quantitative rt-PCR can be performed to answer additional questions about macrophage subpopulation signaling pathways. After its development, this technique paved the way for researchers in neuroscience field to explore the activation of macrophage subpopulation in mouse model of neurologic disease. After watching this video, you should either conduct testing or follow this tradition as macrophage subpopulations by flow cytometry.

Don't forget that working with paraformaldehyde can be extremely hazardous, and that precautions such as using a hood should always be taken while performing this procedure.