The overall goal of this procedure is to isolate immune cells from the ischemic mouse brain for their quantification and phenotypic characterization by multiparametric flow cytometry. Analysis of post-ischemic inflammation in the brain by multicolor flow cytometry provides a deep understanding of the immunological processes implicated in lesion progression, repair, and overall outcome of the stroke. The main advantage of this procedure is the rapid, one-step density gradient centrifugation, which allows the isolation of high numbers of viable immune cells from the ischemic mouse brain.
The application of this technique targets the identification and validation of new immunomodulatory treatment strategies that can be applied in a wider time window of the stroke. Begin by immersing one end of a peristaltic perfusion pump tube into ice-cold calcium-and magnesium-free HBSS. Fix a blunt 23-gauge needle to the other end of the tube.
And switch on the pump to completely fill the tubing with HBSS. Next, place a euthanized mouse dorsally on a dissection board embedded in a plastic tray, and pin the fore and hind paws, stretched as widely as possibly, with 20-gauge needles. Using a pair of straight, one by two teeth forceps to grasp the abdominal skin, make a lateral incision through the integument and abdominal wall with sharp Iris scissors to expose the liver.
Then lift the sternum, and use the blunt blade of a pair of sharp-blunt Iris scissors to incise the diaphragm. Continue to cut the lateral rib cage at both sides in the caudocranial direction, taking care not to injure the lung, heart, and thoracic arteries, and use blunt forceps to fit the skin flap. Pin the flap to the dissection board and carefully separate the heart from connective tissue.
Holding the heart with a blunt-end forceps, then insert the tip of the 23-gauge needle at the end of the perfusion tubing into the apex of the left ventricle, taking care not to injure the intraventricular septum. Using sharp Iris scissors, incise the right atrium, and immediately start the pump, taking care to avoid the formation of bubbles within the pumping. When the liver becomes a light coffee color, use straight surgical scissors to decapitate the mouse just behind the skull.
Then use the Iris scissors to make a midline incision in the scalp. When the skull is visible, place one tip of sharp Iris scissors into the foramen magnum, and make a lateral incision into the bone. Repeat the incision on the other side.
Then use sharp Iris scissors to carefully cut from the same cavity up the midline, towards the nose. Using fine forceps, gently peel the cranial bones from each brain hemisphere. Then lift the brain with a spatula, and use sharp Iris scissors to carefully dissect the cranial nerve fibers that fix it to the skull.
Placing the brain into a 15-milliliter tube, containing 10 milliliters of HBSS with calcium and magnesium on ice. To dissociate the cerebral tissue into a single cell suspension, first, use a clean razor blade to carefully remove the brain stem and cerebullum. Then use another clean razor blade to hemi-sect the brain, cutting each hemisphere along the coronal plane into three pieces of roughly equal size.
Using the plunger end of a five-milliliter syringe, mash the pieces of brain tissue through a 100-micron cell strainer, continuously rinsing the strainer with ice-cold HBSS with calcium and magnesium. Then place the homogenized tissues on ice. When all of the samples have been processed, spin down the cell suspensions and carefully discard the supernatants.
Resuspend the pellets in one milliliter of digestion buffer. Then transfer the cell suspension into a two-milliliter tube and incubate the cells under slow continuous rotation at 37 degrees Celsius. After one hour, sieve the cell suspension through a 70-micron cell strainer and rinse the filter thoroughly with three milliliters of wash buffer containing DNase.
Then wash the strainer with 15 milliliters of DNase-free wash buffer, spin down the cells, and discard the supernatant. To remove the cell debris and myelin, resuspend the cells in five milliliters of room temperature, 25%density gradient medium and transfer the cell suspension into a 15-milliliter tube. Mix the cells in gradient thoroughly with repeated gentle pipetting.
Then spin down the cell solution. At the end of the separation, carefully aspirate the myelin coat and supernatant without disturbing the pellet and resuspend the cells in 10 milliliters of DNase-free wash buffer. Transfer the cells into a 15-milliliter tube and spin them down again, resuspending the pellet in 100 microliters of cold wash buffer.
Then count the number of viable cells by trypan blue exclusion, and store them at four degrees Celsius. Before beginning the immunostaining, incubate the cells at four degrees Celsius for 10 minutes with anti-murine CD26/CD32 Fc receptor blocking reagent to prevent any nonspecific binding. Then add the fluorophore-conjugated primary antibodies of interest at four degrees Celsius for 20 minutes in the dark.
At the end of the incubation, wash the cells in two milliliters of flow cytometry buffer. Spin down. Resuspend the pellet in 200 microliters of flow cytometry buffer, and store the samples at four degrees Celsius until flow cytometric analysis.
To quantify the absolute number of the infiltrating immune cells by bead counting, reverse pipet exactly 40 microliters of flow cytometry and 10 microliters of cell suspension into a counting tube containing a known number of fluorescent beads. Next, incubate the cell and bead suspension with FITC-labeled CD45 antibody at four degrees Celsius for 20 minutes in the dark followed by reverse filling of the counting tube with 200 microliters of flow cytometry buffer. Then immediately run the sample on the flow cytometer, recording the bead, and CD45-positive cell events.
24 hours after middle cerebral artery occlusion, the brain-infiltrating leukocytes can be identified by their CD45-high expression. Within the CD45-high population, the polymorphonuclear neutrophils are identified by their LY-6G expression while the T-lymphocytes exhibit a CD45-high, CD3-positive cell surface phenotype. The remaining CD45-high cells can be further distinguished by their CD19 and CD11b expression.
While the CD11b-positive fraction can be sub-categorized into Ly-6C-high inflammatory monocytes, and a Ly-6C-low population that encompasses the monocyte, macrophage, and dendritic cell populations. In the acute stage of stroke, Ly-6G-positive neutrophils enter the brain rapidly after vessel occlusion. By contrast, frequencies of the CD3-positive T-cells in the ischemic hemisphere decrease compared to sham surgery brains and the contralateral hemisphere.
Within the CD11b-positive population, brain ischemia shifts the balance towards a strong preponderance of Ly-6C-high inflammatory monocytes. Further, the total leukocyte counts in the infarcted hemisphere are significantly increased compared to the contralateral hemisphere and sham surgery brains, 24 hours after middle cerebral artery occlusion. After watching this video, you should have a good understanding of how to isolate immune cells from the ischemic brain by mechanical fragmentation, enzymatic digestion, and density gradient centrifugation, and how to characterize these cells by flow cytometry.
Once mastered, this technique can be completed in four hours, if it's performed properly. While attempting this procedure, it's important to remember that the total cell yield critically depends on thorough dissociation of the brain tissue. Moreover, sufficient transcardial perfusion is necessary to prevent contamination of the brain-infiltrating leukocytes with noninflammatory circulating immune cells.
Following this procedure, other methods like gene expression assays or cytokine profile analysis can be performed to gain additional insights into the functional relevance of distinct immune abscess in the context of ischemic stroke.
