Lung Organoid Co-culture Assay

Lung organoid co-culture assays have been previously described^{46 (link)}. Lung epithelial cells (Epcam⁺CD45^-CD31^-Ter119^-) from control BALB/cJ mice or irradiated mice were FACS sorted and resuspended in 3D organoid media (DMEM/F12 with 10% FBS, 100 U/ml penicillin-streptomycin, and insulin/transferrin/selenium (Merck Sigma-Aldrich)). Cells were mixed with murine normal lung fibroblast (MLg) cells and resuspended in GFR Matrigel at a ratio of 1:1. 100μl of this mixture was pipetted into a 24-well transwell insert with a 0.4 μm pore (Corning). 5,000 epithelial cells and 25,000 MLg cells were seeded in each insert. After incubating for 30 min at 37°C, 500μl organoid media was added to the lower chamber and media changed every other day. Bright-field images were acquired after 14 days using an EVOS microscope (ThermoFisher Scientific), and quantified using FiJi (version 2.0.0-rc-69/1.52r, ImageJ).