Recombinant mouse egf

Organoid assays were performed as previously described (Ng-Blichfeldt et al., 2019 (link)). Pre-treated or pre-exposed MRC-5 fibroblasts were resuspended in a 1:1 ratio (20.000 cells each) with EpCAM⁺ cells in 50 µL DMEM/10% FBS +50 µL growth-factor reduced Matrigel (Corning). Cell suspensions were seeded on 24-transwell inserts and subsequently incubated at 37°C for ∼30 min to enable polymerization of the Matrigel. Organoid cultures were maintained in DMEM/F12 with 5% (vol/vol) FBS, 2 mM L-glutamine, p/s, 1x insulin-transferrin-selenium (Gibco), recombinant mouse EGF (0.025 μg/mL, Sigma), bovine pituitary extract (30 μg/mL, Sigma), and freshly added all-trans retinoic acid (0.01 μM, Sigma) at 37°C with 5% CO₂. Y-27632 (10 μM, Tocris, Oxford, United Kingdom) was added for the first 48 h of culture. To quantify colony-forming efficiency, the total number of organoids per well was counted manually 7 days after seeding using a light microscope at ×20 magnification. Organoid diameter was measured 14 days after seeding with a light microscope connected to NIS-Elements software (Nikon Europe, Netherlands). For immunofluorescence, organoid cultures were fixed with ice-cold acetone/methanol (1:1) for 12 min at −20°C, then non-specific binding sites were blocked in PBS with 5% bovine serum albumin (BSA; Sigma). Samples were stored at 4°C until immunofluorescence analysis.