HBECs were harvested at 15d ALI using 0.05% Trypsin-EDTA (ThermoFisher, 25300054). Cells were then pelleted at 300g for 5 min, resuspended in PBS and filtered through a 20 μm strainer (PluriSelect, 43–50020–03). Cells were counted on a hemocytometer and Optiprep (Sigma-Aldrich, D1556) was added to achieve a final concentration of 15% and 75,000 cells/mL.
C57/BL6 mice from the Jackson Laboratory aged 6–8 wk were used for all studies. Animals were handled in accordance with Novartis Institutes for Biomedical Research Animal Care and Use Committee protocols and regulations. Mice were housed in a temperature- and humidity-controlled animal facility with ad libitum access to food and water and acclimated for at least 3 d before experimental manipulation. For single-cell isolation for scRNA-seq, tracheas were dissected and opened longitudinally in Ham’s F12 (Life Technologies, 11765–054) plus 1% Pen-Strep on ice. Each trachea was individually placed in a 15 mL conical tube with 5 mL of 1.5 mg/mL Pronase (Roche, 10165921001) in Ham’s F12 plus 1% Pen-Strep and incubated for 18h at 4°C. 500 mL FBS was added to inactivate pronase and conical tubes were vigorously inverted to dislodge cells. Each trachea was transferred twice to a 15 mL conical tube containing Ham’s F12 plus 1% Pen-Strep plus 10% FBS and then inverted. Media from each of the three tubes was pooled and cells were pelleted at 400g for 10 min at 4°C. Cells were resuspended in 500 µL DNase (Sigma-Aldrich, DN25), incubated on ice for 5 min and then pelleted at 400g for 10 min at 4°C. Cells were then washed twice in Hams F12 1% Pen-Strep 10% FBS and then resuspended in PBS + 0.02% BSA. Cells were diluted to 90,000 cells/mL in 15% Optiprep + 0.02% BSA in PBS for scRNA-seq.
C57/BL6 mice from the Jackson Laboratory aged 6–8 wk were used for all studies. Animals were handled in accordance with Novartis Institutes for Biomedical Research Animal Care and Use Committee protocols and regulations. Mice were housed in a temperature- and humidity-controlled animal facility with ad libitum access to food and water and acclimated for at least 3 d before experimental manipulation. For single-cell isolation for scRNA-seq, tracheas were dissected and opened longitudinally in Ham’s F12 (Life Technologies, 11765–054) plus 1% Pen-Strep on ice. Each trachea was individually placed in a 15 mL conical tube with 5 mL of 1.5 mg/mL Pronase (Roche, 10165921001) in Ham’s F12 plus 1% Pen-Strep and incubated for 18h at 4°C. 500 mL FBS was added to inactivate pronase and conical tubes were vigorously inverted to dislodge cells. Each trachea was transferred twice to a 15 mL conical tube containing Ham’s F12 plus 1% Pen-Strep plus 10% FBS and then inverted. Media from each of the three tubes was pooled and cells were pelleted at 400g for 10 min at 4°C. Cells were resuspended in 500 µL DNase (Sigma-Aldrich, DN25), incubated on ice for 5 min and then pelleted at 400g for 10 min at 4°C. Cells were then washed twice in Hams F12 1% Pen-Strep 10% FBS and then resuspended in PBS + 0.02% BSA. Cells were diluted to 90,000 cells/mL in 15% Optiprep + 0.02% BSA in PBS for scRNA-seq.
