DMEM/F-12, no phenol red

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Seed unsorted or sorted lung epithelial cells (1–2 × 104 cells in 50 μl Matrigel/well) Change medium every 3 days.

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Seed unsorted or sorted lung epithelial cells (1–2 × 104 cells in 50 μl Matrigel/well) Change medium every 3 days.

Publication protocol

Lung Organoid Culture in 3D Matrigel: The unsorted or sorted lung epithelial cells were resuspended in Matrigel and plated into a Matrigel-coated 24-well plate in domes (1–2 × 104 cells in 50 μl Matrigel/well). The plate was incubated at 37°C for 30–45 min before adding 1 ml of medium per well. The cells were incubated in a humidified atmosphere of a cell culture incubator (37°C, 5% CO2). The medium was changed every 3 days. The media used were the following: Lungosphere medium [1 × B-27 without vitamin A, 100 U/ml penicillin, 100 μg/ml streptomycin, in phenol red-free DMEM/F12], supplemented with EGF (20 ng/ml) and/or FGFs [1 or 2 nM FGF2-STAB, FGF2-wt, murine FGF7 (#450-60, Peprotech), murine FGF9 (#450-30, Peprotech), or human FGF10 (#100-26, Peprotech)] as needed according to experiment, and 10 μM Y-27632 (only for the first 3 days of culture); a WNT lung organoid medium (Lee et al., 2017): 50 ng/ml murine WNT3A (#315-20, Peprotech) or 50% WNT3A-conditioned medium,100 ng/ml murine Noggin (#250-38, Peprotech), 500 ng/ml human R-spondin 1 (#120-44, Peprotech), and 40 ng/ml EGF in lungosphere medium, with or without 40 ng/ml FGF10. Organoid forming efficiency (OFE,%) was calculated as (number of organoids formed)/(number of cells seeded) × 100. Organoid size was measured from organoid photographs using ImageJ (NIH) as the area occupied by the organoid. lungosphere medium [1 × B-27 without vitamin A, 100 U/ml penicillin, 100 μg/ml streptomycin (all Thermo Fisher Scientific), 4 μg/ml heparin (Sigma/Merck), 20 ng/ml murine EGF (#315-09, Peprotech), 10 ng/ml FGF2-STAB (thermostable FGF2 based on human FGF2 sequence; Enantis), or 10 ng/ml human FGF2-wt (#100-18C, Peprotech), 10 μM Y-27632 (Merck) in phenol red-free DMEM/F12 (Thermo Fisher Scientific)]

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