This protocol can be used to investigate alterations in the pulmonary environment following ischemic stroke. This method allows for the identification of 13 lung immune cell types and 13 different cytokines and chemokines in the same mouse via flow cytometric analysis. This method has been utilized to assess alterations in the pulmonary environment following ischemic stroke.
However, it can also serve to investigate changes during allergic response or infection. To perform whole body transcardial perfusion, confirm a lack of response to pedal reflect in an anesthetized mouse and secure the mouse to a surgical platform. Wet the fur with 70%ethanol to reduce fur distribution into the body cavity and make a vertical midline skin incision to expose the intact peritoneum stopping the incision as soon as the thoracic cavity has been reached.
Use fine dissection scissors to make a midline incision of the peritoneum stopping at the thoracic cavity prior and use forceps to grasp the distal end of the sternum. Use the scissors to cut through the diaphragm without damaging the heart, lungs, or vasculature. When the underlying tissues are visible, make an incision through the rib cage and secure the sides of the ribs so that the heart and lungs are fully exposed.
Carefully make a small incision in the right atrium near the aortic arch to serve as the outflow for the perfusion and quickly but gently insert a 25 gauge needle into the distal superior surface of the left ventricle. Once the needle is in place gently flush 10 milliliters of cold PBS into the heart. The tissue should begin to clear of blood.
When the entire volume has been delivered, perfuse a second 10 milliliter aliquot of cold PBS until the liver exhibits a light brown appearance and the lungs have transitioned from a reddish pink to a mostly white in color. Using forceps and fine dissection scissors, carefully remove all of the surrounding tissues before separating the lung lobes and placing them into a Petri dish containing one to two milliliters of cold lung cell, medium on ice. To prepare the lung tissue for multiplex bead arrays add 200 microliters of cold homogenization buffer to a two milliliter tube containing three sterile 2.3 milliliters Zirconia/Silica beads, and weigh the lung lobe of interest.
Transfer 50 to 100 milligrams of lung tissue to the pre-chilled tube of beads and homogenization buffer and homogenize the lobe using a bead based homogenizer at 4, 000 rotations per minute for at least two minutes. When the tissue has been completely homogenized, centrifuge the tube to sediment the tissue and transfer the super natant to a pre-chilled 1.5 milliliter micro tube on ice. For lung tissue dissociation carefully decant the lung cell medium from the remaining lung lobe and insert a 25 gauge needle into the tissue.
Inject 200 to 250 microliter volumes of dissociation buffer into each lung lobe to inflate the lungs until each of the lobes have been injected two to three times. After a two minute incubation at room temperature use fine dissection scissors to mince the lungs into small pieces and transfer the tissue solution into 15 milliliter chronicle tube. Add six milliliters of additional dissociation buffer to the sample and vortex vigorously for one minute.
Next, incubate the tissue pieces for 45 minutes at 37 degrees Celsius vortexing every seven to eight minutes. At the end of the incubation, vortex the sample vigorously for one minute and pass the sample through a 100 micron cell strainer to remove any residual undesired connective and interstitial tissue. Collect the tissue by centrifugation and resuspend to the pellet with five milliliters of cold lung medium.
Centrifuge the sample again and resuspend the pellet in one milliliter of cold lung medium with gentle vortexing. Then, filter the cells through a 100 micron strainer before counting. To evaluate the immune cell populations within the isolated lung tissue, seed one to two times 10 to the sixth power cells antibody set into the appropriate number of wells with a 96 well round bottom plate and dispose the cells to the bottom of the plate by centrifugation.
Add 25 microliters of the appropriate antibody cocktail in cold FACS buffer to each well and mix with gentle pipe petting. After 20 minutes, resuspend the cells in 100 microliters of cold FACS buffer per well and transfer the samples to individual five milliliter polystyrene round bottom tubes containing 150 microliters of additional FACS buffer per tube. Then, immediately analyze the samples on a flow cytometer according to standard flow cytometric analysis protocols.
In this representative analysis, 13 different populations of immune cells were identified in the lungs using three sets of five to seven antibody combinations. Dead cells were excluded using a live dead stain and the different immune cell types were distinguished using the antibodies and markers listed in the table. Alveolar and interstitial macrophages, CD103 positive and CD11B positive dendritic cells and eosinophils were identified in set one.
Pro-inflammatory monocytes and neutrophils were identified in set two and CD4 positive and CD8 positive T cells, B cells, plasmacytoid dendritic cells, NK cells, and NKT cells were identified in set three. The total number of viable cells percentage of CD45 positive cells and total number of CD45 positive cells obtained were similar between cell populations that were isolated as demonstrated and cell populations that were isolated using a commercially available tissue dissociator. In addition, the percentage of cell death among CD45 positive cells was less than 10%for both protocols, suggesting that the demonstrated protocol facilitates a high yield, high cell quality recovery without the aid of an automated tissue dissociator.
Further, using a commercially available multiplex assay coupled with flow cytometric analysis allows the concentration of chemokine in each sample per milligram of tissue to be determined. The collagenase D concentration, incubation time and temperature used during the single cell isolation critically impacts the immune cell recovery.
