Inject one to three milligrams of fluorophore-conjugated anti-mouse CD45 antibody diluted in 100 milliliters of normal saline intravenously into each anesthetized mouse. Begin by placing four milliliters of ice-cold HEPES buffer prepared in a specialized tissue dissociator tube on ice for each mouse. After euthanizing the mouse, place the resected lungs in their respective tissue dissociator tube, chilled on ice.
Place the tubes onto the automated tissue dissociator and run the first preset program for lung tissue. Then, add 500 microliters of liberase stock solution and 500 microliters of aminoguanidine stock solution to each tube containing the dissociated lung tissue. Incubate it on a nutating mixer for 30 minutes at 37 degrees Celsius.
Afterward, place the tubes back on the dissociator and run the second preset program for lung tissue. Now pour the single-cell suspension from the tissue dissociator tube through a 100-micrometer cell strainer into a 50-milliliter conical tube. Rinse the tissue dissociator tube with five milliliters of complete EHAA and pass through the strainer into the 50-milliliter tube.
After removing the strainer, raise the volume of the cell suspension to 50 milliliters with complete EHAA. Instead of using specialized tissue dissociator tubes, place the resected lungs in their respective 1.5 milliliter-microfuge tubes chilled on ice. Once all the lungs have been harvested, transfer each lung to a fresh microfuge tube without any cell media.
Using scissors, cut the lungs into small fragments within the microfuge tube. Add one milliliter of liberase TM and DNase I digestion cocktail to each tube. Incubate for 30 minutes at 37 degrees Celsius in a water bath.
Then, pour the sample over a 100-micrometer cell strainer placed on top of a 50-milliliter conical tube. Pipette out remaining sample onto the strainer. Mash the tissue against the mesh with the rubber end of the plunger from a sterile one-milliliter syringe.
Rinse the strainer with cold sorter buffer, collecting a final volume of seven milliliters in the tube. Whether using the automated or manual dissociation method, centrifuge the cell suspension obtained. Carefully aspirate the supernatant, leaving behind approximately 100 microliters of supernatant and the cell pellet.
Finally, add approximately 100 microliters of Fc block solution to bring the total volume up to 200 microliters and vortex vigorously to resuspend the pellet.
