Isolation and Culture of Human Lung Cells

An immune-cell-depleted single-cell suspension of peripheral human lungs was obtained as described previously^{65 (link)}. Immune-cell-depleted single-cell suspension was stained with the following antibodies: anti-DRAQ7 (Cell Signaling Technologies, 7406S, 1:200) for dead cell exclusion, mouse anti-human CD45 APC-Cy7 (BioLegend, 304014, 1:200), mouse anti-human anti-CD11b-APC-Cy7 (BD, 557754, 1:200), mouse anti-human CD31 APC-Cy7 (BioLegend, 303120, 1:200), mouse anti-human CD326 PE (BioLegend, 324206, 1:200), mouse IgM anti-human HTII-280 (Terrace Biotech, 303118, 1:200), mouse anti-human NGFR-APC (BioLegend, 345108, 1:100) and mouse anti-human CEACAM6-PE (Thermo Fisher Scientific, 12-0667-42, 1:100). Finally, goat anti-mouse IgM Alexa Fluor 488 (Thermo Fisher Scientific, A-21042, 1:1,000) was added to label HTII-280⁺ cells. Cells were sorted using the BD FACSAria II (BD Bioscience) according to the manufacturer’s instructions. Next, 10,000 sorted cells were resuspended in 40 μl of growth-factor-reduced Matrigel (Corning, 354230) and placed as droplets directly in cell culture plates. Cell–Matrigel suspension was allowed to polymerize at 37 °C for 20 min followed by addition of the appropriate cell culture medium.