Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

We study lung repair after viral infection. We aim to understand the mechanisms by which immune cells mediate epithelial repair and how remodeling after viral injury and subsequent repair can impact future disease pathologies. This protocol addresses the incompatibility of lung digests optimized for either epithelial and stromal cells or immune cells.

Our research requires isolating viable cells from both immune and non-immune compartments. Furthermore, we put forward a gating strategy for epithelial and macrophage subsets, which we hope will benefit the field. Our laboratory investigates the crosstalk between immune and epithelial cells in the respiratory tract under steady state conditions and in response to environmental challenges.

Our ultimate goal is to determine how best to modulate this communication to enhance tissue repair and prevent disease development. To begin, spray the mouse with 70%ethanol. Using fine surgical scissors and number seven forceps, make an incision into the abdomen and cut laterally through the peritoneum.

Make a small cut into the diaphragm to release the vacuum. Then, cut the diaphragm and lower ribcage out of the body cavity to expose the lungs and heart. Using a 10 milliliter syringe fitted with a 27 gauge needle, perfuse the lungs with five milliliters of two millimolar EDTA in DPBS.

Cut vertically through the sternum and along the sides to open the ribcage and expose the trachea. Use fine scissors and number seven forceps to remove as much muscle and connective tissue as possible without puncturing the trachea or lungs. Next, wrap the trachea with a 2/0 suture, positioning it as if to tie it off, but do not tighten.

Make a lateral nick in the trachea and insert the catheter. Then, pull the suture tight to secure the catheter in place. Inflate the lungs with one milliliter of DPBS using a syringe.

Then, slowly pull back on the syringe to pull out the DPBS. Reinflate the lungs with the same DPBS, and again, pull back on the syringe to collect the bronchoalveolar lavage fluid, or BAL. Disconnect the syringe from the catheter while leaving the catheter inserted.

Dispense the collected valve into a micro centrifuge tube. Next, fill the same one milliliter syringe with one milliliter of dispase. Attach the syringe to the catheter and inflate the lungs with dispase.

While removing the catheter, tighten the suture around the trachea to prevent leakage of dispase. Using scissors, cut laterally through the trachea. Remove the lungs from the body cavity by cutting through the connective tissue along the back of the ribcage while pulling the lungs and trachea upward.

Then, remove the heart from the lungs. Place the lungs in five milliliters of DPBS on ice. Centrifuge valve at 400 G for five minutes at four degrees Celsius.

With a pipette, aspirate the supernatant and resuspend the pellet in 100 microliters of RPMI. To begin, dissect the five lobes of the lungs harvested from the mouse and discard the connective tissue. Chop the dissected lung tissue with scissors for one minute on a glass slide.

Using dissection scissors, transfer the chopped lung tissue into a 50 milliliter centrifuge tube. Then, add three milliliters of digest mix and pipette the lung digest mixture up and down two to three times. Finally, add the cell pellet recovered from BAL resuspended in RPMI.

Place the tube at a 45 degree angle in an orbital shaker set to 140 RPM at 37 degrees Celsius for 40 minutes. After incubation, remove the tube from the shaker and place it on ice. Pipette the lung digest mixture up and down five to six times.

Filter the mixture through a 70 micrometer cell strainer into a new 50 milliliter tube. Wash any remaining cells through the filter and neutralize the enzymes with 10 milliliters of 5%FBS in the RPMI medium. Centrifuge the sample at 600 G for five minutes at four degrees Celsius.

Aspirate the supernatant using a vacuum aspirator. Resuspend the cell pellet in one milliliter of ACK lysis buffer and incubate for three minutes at room temperature to lyse red blood cells. Then, add nine milliliters of PBS and pipette up and down to mix the solution.

Again, centrifuge the sample and use a vacuum aspirator to remove the supernatant, then resuspend the cell pellet in one milliliter of fluorescence-activated cell sorting buffer. Filter the suspension through a 64 micrometer mesh into a microcentrifuge tube for flow cytometry analysis.