Isolation of Lung Retinoid-Containing Cells by Cell Sorting

Our research focuses on understanding the physiological role of vitamin A and its metabolites, collectively term retinoids, in maintaining the functional health of the adult lung.

Due to the lung's highly specialized and heterogeneous cellular composition, single-cell techniques such as specific cell labeling, sorting and sequencing are essential for studying retinoid mediated cellular communication in lung health and disease.

Our research has unveiled the previously unsuspected complexity of retinoid metabolism and signaling in the adult lung, and its critical role in protecting the lung during injuries.

Our protocol enables the isolation of specific lung cell populations based on the endogenous retinoid autofluorescence, and can be easily integrated with fluorochrome-conjugated antibody stain in all fluorescent protein expression.

Our studies are paradigm shifting for understanding the mechanisms of vitamin A metabolism and its actions. The outcomes of these studies can and will impact current strategies, including nutritional interventions aimed at alleviating lung disease.

[Presenter] To begin, perform the toe pinch test on an anesthetized mouse to check the level of unconsciousness. Remove loose hair and visible dirt from the surgical site. Then, wipe the area clean using 70% alcohol for disinfection. Using sterile surgical tools, open the abdominal and chest cavities. Cut the ribs and diaphragm to expose the lungs and heart. Then, cut the inferior vena cava. Apply an absorbent pad to absorb released blood. Perfuse the lungs in-situ through the heart's right ventricle with 5 milliliters of sterile HBSS without calcium and magnesium. Verify successful perfusion by observing the lung tissue turning white. Perfuse the lungs again using another 10-milliliter syringe filled with sterile HBSS containing enzymes, calcium, and magnesium. Now, remove the lungs and transfer them into a cell culture dish containing 2 milliliters of sterile HBSS with calcium and magnesium. Under a laminar hood, rinse the lungs and mince them into small pieces with a sterile surgical blade. Add 5 milliliters of enzyme HBSS containing calcium and magnesium to the minced lungs. Then, use a 10-milliliter serological pipette to transfer the suspension into a 15-milliliter tube. Wash the cell culture dish with an additional 5 milliliters of enzyme containing HBSS. Transfer the wash solution to the 15-milliliter tube. Incubate the minced lung tissue in the enzyme containing HBSS on a rotating shaker at 37 degrees Celsius for 45 minutes. Every 15 minutes, pipette the minced tissue 10 times through a 10-milliliter serological pipette inside the laminar hood to enhance dissociation. Do this three times. Pass the resulting cell suspension through a 100-micrometer strainer into a 50-milliliter tube containing 20 milliliters of cold live cell imaging solution containing less than 5% FBS. Centrifuge the cells at 500 g for 10 minutes at 4 degrees Celsius. After aspirating the supernatant, resuspend the cell pellet in 1 milliliter of red blood cell lysing buffer and incubate. When the erythrocytes have lysed, add 20 milliliters of cold live cell imaging solution to the suspension before centrifuging again. Aspirate the supernatant and resuspend the cell pellet in 20 milliliters of cold live cell imaging solution. Pass the resultant cell suspension through a 40-micrometer cell strainer into a 50-milliliter tube containing 20 milliliters of cold live cell imaging solution. Centrifuge the cell suspension as before to collect the cells. Finally, aspirate out the supernatant and resuspend the cell pellet in 10 milliliters of cold live cell imaging solution. Count the cells under a microscope using a hemocytometer and adjust the concentration to approximately 5 to 10 million cells per milliliter. Take an aliquot of the prepared cell suspension and set it aside as an unstained gating control. Add viability dye cytoscreen at a 1 to 1000 dilution to the remaining cell suspension to achieve a final concentration of 30 nanomolar. Next, attach a filter to a polystyrene 5-milliliter collecting tube. Pass the suspension through the filter. Perform fluorescence-activated cell sorting to isolate live individual retinoid-containing cells based on their emission at 455 nanometers. Perform sequential singlet discrimination using a forward scatter height versus forward scatter area plot. Exclude dead cells based on scatter characteristics and cytoscreen staining. Gate, sort, and collect singled live retinoid-containing cells based on their emission at 455 nanometers upon excitation at 350 nanometers. Finally, perform FACS data analysis using flow cytometry software. A distinct population of live single cells with high autofluorescence at 455 nanometers was detected in the lung cell suspensions wild-type mice. The same autofluorescent population was absent in Lrat knockout lung cell suspensions. A subpopulation of tdTomato positive fibroblasts was successfully isolated using a Cre-inducible labeling system, allowing further separation of retinoid-containing cells from this cell population based on autofluorescence. The presence of tdTomato positive lung fibroblasts was confirmed using fluorescence microscopy showing red-labeled cells. Retinoid presence in sorted fibroblasts was validated using HPLC, identifying retinol and retinol esters. Lipid droplets were detected in retinoid-containing fibroblasts.