Protease type xiv from s griseus

Single-cell analysis was performed at the indicated days of culture at the air-liquid interface. To obtain a single-cell suspension, cells were incubated with 0.1% protease type XIV from S. griseus (Sigma-Aldrich) in supplemented HBSS for 4 h at 4°C. Cells were gently detached from Transwells by pipetting and then transferred to a microtube. Fifty units of DNase I (EN0523 Thermo Fisher Scientific) per 250 µl were directly added and cells were further incubated at room temperature for 10 min. Cells were centrifuged (150 g for 5 min) and resuspended in 500 µl supplemented HBSS containing 10% FCS, centrifuged again (150 g for 5 min) and resuspended in 500 µl HBSS before being mechanically dissociated through a 26 G syringe (four times). Finally, cell suspensions were filtered through a 40 µm porosity Flowmi Cell Strainer (Bel-Art), centrifuged (150 g for 5 min) and resuspended in 500 µl of ice-cold HBSS. Cell concentration measurements were performed with a Scepter 2.0 Cell Counter (Millipore) and Countess automated cell counter (Thermo Fisher Scientific). Cell viability was checked with a Countess automated cell counter (Thermo Fisher Scientific). All steps except the DNase I incubation were performed on ice. For cell capture using the 10× genomics device, the cell concentration was adjusted to 300 cells/µl in HBSS, aiming to capture 1500 cells for HAECs and 5000 cells for MTECs.

Fully differentiated HAECs were dissociated by incubation with 0.1% protease type XIV from S. griseus (Sigma-Aldrich) in HBSS (Hanks’ balanced salts) for 4 h at 4 °C. Cells were gently detached from the Transwells® by pipetting and then transferred to a microtube. Cells were then cytocentrifuged at 72×g for 8 min onto SuperFrostPlus slides using a Shandon Cytospin 3 cytocentrifuge. Slides were fixed for 10 min in methanol at −20 °C for Centrin2 and ZO1 assays, and for 10 min in 4% paraformaldehyde at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 10 min for acetylated-α-tubulin assays. Cells were blocked with 3% BSA in PBS for 30 min. The incubation with primary antibodies was carried out at room temperature for 2 h. Then, mouse and rabbit secondary antibodies from the Duolink® Red kit (Sigma-Aldrich) were applied and slides were processed according to manufacturer’s instructions. Images were acquired using the Olympus Fv10i confocal imaging systems with ×60 oil immersion objective and Alexa 647 detection parameters.