Because the lung hosts a very complex and unique immune system, several gating strategies for identification of lung immune cells have been developed and reported. The existence of several different approaches makes it difficult to compare results generated by different laboratories. Our gating strategy provides a comprehensive and reproducible way to identify up to 12 different pulmonary myeloid and non-myeloid immune populations using nine markers.
The present protocol describes characterization and identification of lung immune populations under steady-state conditions. However, this protocol can be employed to identify changes of these cell populations in various disease models, where it can help identify disease-specific changes of the lung immune landscape. Investigators who perform this technique for the first time should pay attention to the digestion conditions and reagents, as they make a big difference in the release of immune cells from the lung tissue.
Begin by preparing the euthanized animal for surgery. Stabilize the mouse dorsally by using needles or tape on the four extremities, then use 70%ethanol to sanitize the skin of the ventral area. Make an incision from the neck to the abdomen.
Remove the skin from the thoracic area, along with the ribs and the sternum. Flush the lungs by injecting 10 milliliters of cold PBS into the right ventricle using an 18 to 21-gauge needle until they become completely white. Then, remove the thymus and heart without touching the lungs.
Detach the lungs from the surrounding tissues and transfer them into a tube containing cold BSA buffer. Transfer the lung to a Petri dish, mince it using two fine scalpels, and then place it into a 50-milliliter conical tube. Add five milliliters of digestion buffer to wash the plate.
Secure the lid of the tube and digest the lung for 30 minutes on an orbital shaker at a speed of 150 rotations per minute at 37 degrees Celsius. Stop the reaction by adding 10 milliliters of cold BSA buffer. After digestion, mix and dissolve the lung pieces using an 18-gauge needle.
Place a 70-micrometer filter strainer on top of a new 50-milliliter conical tube and transfer the digested lung mixture into the strainer. Use the rubber side of a 10-milliliter syringe plunger to smash the remaining lung pieces on the filter and wash it with BSA buffer. Centrifuge the single-cell suspension at 350 G for eight minutes at seven degrees Celsius.
Discard the supernatant and resuspend the cells in one milliliter of ACK lysis buffer. Mix the suspension using a one-milliliter pipette and incubate it for 90 seconds at room temperature. Add 10 milliliters of cold BSA buffer to the reaction mixture and centrifuge it at 350 G for seven minutes at four degrees Celsius.
Discard the supernatant, resuspend the pellet in staining buffer, and count the cells using a hemocytometer. Resuspend the cells at a concentration of 5 million cells per milliliter for surface staining. Transfer 1 million cells in 200 microliters per well into a 96-well plate and centrifuge the plate at 350 G for seven minutes at four degrees Celsius.
Prepare a flow cytometry block solution by diluting anti-1632 antibody in staining buffer. Resuspend the cells in 50 microliters of the flow cytometry block solution. Incubate the suspension for 15 to 20 minutes at four degrees for Celsius or on ice.
Then, add 150 microliters of staining buffer to the plate and centrifuge it 350 G for five minutes at four degrees Celsius. Prepare surface antibody solution by diluting the surface antibodies in staining buffer. Resuspend the cells in 50 microliters of the surface antibody solution and incubate the plate at four degrees Celsius in the dark.
Then, wash the cells twice with staining buffer. Prepare the fixation and permeabilization buffer by mixing three parts fixation and permeabilization diluent and one part staining buffer. Resuspend the cells in 50 microliters of the preprepared buffer per well of the 96-well plate and incubate them for 20 to 25 minutes at four degrees Celsius in the dark.
Dilute the permeabilization buffer with purified deionized water to prepare one times permeabilization buffer and use it to wash the cells. Prepare an intracellular antibody solution by diluting with one milliliter of permeabilization buffer. Resuspend the cells in 50 microliters of the diluted intracellular antibody solution per well of the 96-well plate and incubate for 40 minutes at four degrees Celsius in the dark.
Wash the cells with permeabilization buffer, then with staining buffer. After the final wash, resuspend the cells in 200 microliters of staining buffer. Acquire a minimum of 1.5 million cells per sample on the flow cytometer.
After a successful surgery and appropriate postoperative procedure, the debris and doublets were excluded through a gating strategy. Immune cells were identified using the CD45+hematopoietic marker via flow cytometry. The cells were distinguished as live or dead.
The immune lung cells were categorized into three groups using anti-GR-1 and anti-CD68 antibodies. Neutrophils were identified and verified using Ly6G as a unique marker. The CD45+population also contains natural killer cells, T cells, and B cells.
These cells were stained and verified by natural killer 1.1, CD3, and B220 antibodies. The bronchoalveolar lavage identified eosinophils which were positive for the CD11b marker, and alveolar macrophages that did not express high levels of CD11b. CD45+dendritic cells were found and confirmed by CD24 expression.
These cells showed either a positive or negative response towards CD103 and CD11b markers. CD103 negative cells were classified as conventional and monocyte-derived dendritic cells based on CD64 expression. the monocyte-derived dendritic cells were low positive for the CD24 dendritic marker, and positive for the CD64 pan-macrophage marker.
Interstitial macrophages with classical and non-classical monocytes were distinguished based on CD64 and GR-1 expression. These cells showed positive expression for CX3C chemokine receptor one. The most important steps in this procedure are proper tissue harvesting, digestion, and preparation of single-cell suspension, and gating on live cells and singlets.
In addition to characterizing immune cells of the lung by flow cytometry, cell culture and functional assays can be performed in isolated cell populations. This technique can pave the way for researchers to explore new questions in diseases of the lung, such as infections, autoimmune diseases, and cancer.
