Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

All animal experiments must conform to according standards for animal care (e.g., the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, NIH Publication No. 85-23, revised 1996) and have to be approved by the appropriate state authority.

1. Preparation of Reagents

1. Digestion buffer (1 ml per hemisphere): Dissolve a standardized mixture of purified digestive enzymes, e.g., Liberase with low thermolysin concentration (TL) to a concentration of 2U/ml in Hanks Balanced Salt Solution (HBSS) containing calcium (Ca) and magnesium (Mg)
2. Washing buffer with DNAse: Dissolve DNAse I to a concentration of 666U/ml in HBSS (Ca/Mg free) containing 10% of fetal calf serum (FCS)
3. Washing buffer without DNAse: HBSS (Ca/Mg free) containing 10% of FCS
4. Density gradient medium (5 ml per hemisphere): prepare stock isotonic density gradient medium (SIP; 100%) by mixing nine parts of density gradient medium with one part 1.5 M sodium chloride. Dilute SIP to 25% density by adding an appropriate volume of HBSS (Ca/Mg free) containing 3% of FCS
5. Flow cytometry (FC) buffer: Prepare phosphate buffered saline (PBS) containing 3% of FCS

2. Transient Middle Cerebral Artery Occlusion

NOTE: Transient middle cerebral artery occlusion (MCAO) via the intraluminal suture technique was performed as described previously¹⁸ in 12-weeks old male C57BL/6 mice.

1. Briefly, anesthetize mice with 2.0% isoflurane in 100% oxygen. Maintain the body temperature at 36.5 °C ± 0.5 °C by a feedback-controlled heating device.
2. After ligation of the common carotid artery and the external carotid artery, introduce a standardized silicon-rubber coated monofilament in the common carotid artery and advance it to the origin of the middle cerebral artery.
3. After 45 min remove the filament to allow reperfusion. In sham-operated animals, immediately withdraw the filament after occluding the middle cerebral artery to avoid ischemia.

3. Transcardial Perfusion and Brain Dissection

1. Prepare a peristaltic perfusion pump by immersing one end of the tubing into ice-cold HBSS (Ca/Mg free). Fix a blunt 23 G needle to the other end of the perfusion tube and switch on the pump to completely fill the tubing with HBSS (Ca/Mg free).
2. Deeply anesthetize mouse with 4% isoflurane and euthanize by CO₂ inhalation.
3. Place mouse dorsally on a dissection board embedded in a plastic tray. Spread fore and hind paws as wide as possible and fix them on the dissection board with 20 G needles.
4. Grab abdominal skin with a straight 1 x 2 teeth forceps and use sharp iris scissors to make a lateral incision through the integument and abdominal wall and expose the liver.
5. Lift the sternum and incise the diaphragm with the blunt blade of sharp/blunt iris scissors. Continue to cut the lateral rib cage at both sides in caudocranial direction. Be careful not to injure the lung, the heart and the thoracic arteries.
6. Lift the skin flap with a blunt forceps and pin it on the dissection board. Use blunt-end forceps and scissors to carefully separate the heart from connective tissue.
7. Hold heart with a blunt-end forceps and insert the tip of the blunt 23 G needle with the attached perfusion tube into the apex of the left ventricle. Note: Do not insert the needle too far into the left ventricle to avoid injury of the interventricular septum. If necessary, fix the cannula in place with a straight sharp forceps.
8. Incise the right atrium with sharp iris scissors and immediately turn on the pump (flow rate: 8 – 10 ml/min).
9. Continue perfusion until the liver shows a light coffee color (~30 ml of HBSS). Throughout the perfusion, carefully avoid any air bubble formation in the tubing.
10. Decapitate the mouse with straight surgical scissors just behind the skull. Use iris scissors to make a midline incision of the scalp to expose the skull.
11. Place one tip of sharp iris scissors into the foramen magnum and cut laterally into the skull. Repeat for the other side.
12. Use sharp iris scissors to carefully cut from the same cavity up the midline towards the nose. Try to keep the end of the scissors as superficial as possible to avoid injury of the brain.
13. Use fine forceps to gently peel the cranial bones from each brain hemisphere. Note: Infarcted tissue may adhere to the meninges or the skull; make sure not to lose brain tissue due to incautious dissection
14. Lift the brain with a spatula and use sharp iris scissors to carefully dissect cranial nerve fibers that fix it to the skull. Place the brain into a 15 ml tube filled with 10 ml of HBSS (+Ca/Mg) and keep it shortly on ice.

4. Dissociation of Cerebral Tissue into Single Cell Suspensions

1. Carefully remove brain stem and cerebellum with a clean razor blade. Use a clean razor blade to hemisect the brain and cut each hemisphere along the coronal plane into three pieces of roughly equal size.
2. Mince dissected tissue of each hemisphere through a 100 µm cell strainer using the plunger end of a 5 ml syringe. Continuously rinse the cell strainer with ice-cold HBSS (+Ca/Mg). Store homogenized samples on ice.
3. Centrifuge at 286 x g and 4° C for 5 min, carefully discard the supernatant. Resuspend the pellet in 1 ml of digestion buffer and transfer the suspension into a 2 ml tube. Incubate suspension under slow continuous rotation at 37 °C for 1 hr.
4. Sieve cell suspension through a 70 µm cell strainer and rinse thoroughly with 3 ml of washing buffer containing DNAse followed by 15 ml of DNAse-free washing buffer. Centrifuge at 286 x g and 18 °C for 5 min and discard the supernatant. Note: Note: The use of DNAse eliminates DNA mucus caused by cell lysis which might lead to cell aggregation compromising cell survival.

5. Density Gradient Centrifugation for Removal of Myelin and Cell Debris

1. Resuspend cell pellet in 5 ml of 25% density gradient medium and pipette the suspension to a 15 ml tube. Mix thoroughly by repeated and gentle pipetting avoiding bubble formation. Note: density gradient medium should be used at RT to prevent cell clumping.
2. Centrifuge at 521 x g and 18 °C for 20 min. Use lowest acceleration profile of the rotor and allow the rotor to stop without brake. Gently remove the tubes from the centrifuge without shaking. Carefully aspirate the myelin coat and the supernatant while preserving the cell pellet at the bottom of tube. Note: Make sure to remove the entire myelin coat as any leftover will impair flow cytometric analysis.
3. Resuspend the pellet in 10 ml of DNAse free washing buffer and pipette the suspension to a new 15 ml tube. Note:This washing step is crucial since sufficient removal of density gradient medium significantly contributes to the quantity and quality of the final cell sample.
4. Centrifuge again at 286 x g and 10 °C for 5 min and discard the supernatant. Resuspend cells in 100 µl of cold washing buffer and determine cell counts and viability by trypan blue exclusion in a hemocytometer. Store samples at 4 °C and further process them quickly.

6. Antibody Staining for Flow Cytometric Analysis

1. Prior to antibody labeling, incubate the cell suspension at 4 °C for 10 min with anti-murine CD16/CD32 FC-Receptor blocking reagent (final concentration of 2.5 µg/ml, dilution factor 1:200) to prevent unspecific binding.
2. Add fluorophore-conjugated primary antibodies at the appropriate concentration (as indicated in Table 1) to the cell suspension and incubate at 4 °C for 20 min in the dark. Note: Control samples are not stained with primary antibodies, but otherwise treated the same.
3. Wash cells with 2 ml of FC buffer and centrifuge at 350 x g and 10 °C for 7 min.Carefully aspirate the supernatant and resuspend cell pellet in 200 µl of FC buffer. Store samples temporarily at 4 °C in the dark until flow cytometric analysis.

7. Absolute Quantification by Counting Beads

1. Reversely pipette 40 µl of FC buffer and 10 µl of the cell suspension into a counting tube containing a known number of fluorescent beads. Note: Pay attention that pipettes are calibrated to deliver exactly 10 µl of sample as subsequent calculation of leukocyte counts refers to this volume.
2. Incubate cell suspension with FITC-labeled CD45 antibody (final concentration of 5 µg/ml, dilution factor 1:100) at 4 °C for 20 min in the dark.
3. Reversely fill the counting tube with 200 µl of FC buffer without any washing step and directly record CD45+ cells in the flow cytometer.

8. Flow Cytometric Acquisition

Note: To analyze the proposed antibody panel, the appropriate cytometer has to be equipped with a blue (488 nm), red (635 nm) and violet (405 nm) laser and filters for FITC, PE, PerCP Cy5.5, PECy7, APC-Cy7 and AmCyan.

1. Adjust forward (FSC) and sideward (SSC) scatter by using unstained cells from an ischemic hemisphere. Discriminate doublets from single cells in a dot plot showing the area and height of FSC.
2. Display all single cells in single-parameter histograms for each fluorescence channel used and adjust the photomultiplier tube (PMT) voltages so that single cells are displayed in the far left of the histogram.
3. Use a CD45-FITC single stained sample to check scatter and PMT settings for the cells of interest by backgating CD45^high immune cells in each histogram and scatter plot. Note: Adapt PMT voltages of FITC so that CD45 negative, intermediate (int) and high expressing cells can be differentiated from each other.
4. Use antibody captured compensation beads to perform multi-color compensation. Set gates for microglia (CD45^int) and leukocytes (CD45^high) and subsequently define leukocyte subpopulations according to the gating strategy displayed in Figure 1.