DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Use 24-well transwell filter inserts with 0.4um pore (Corning) in a 24-well tissue culture plate Seed 2,000 cells for primary organoid (1°) formation

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Use 24-well transwell filter inserts with 0.4um pore (Corning) in a 24-well tissue culture plate Seed 2,000 cells for primary organoid (1°) formation

Publication protocol

Epithelial cell isolation and 3D organoid assay: Primary lung epithelial cells were isolated from 8–10 week old β-actin-EGFP mice or β-actin-dsRed mice, as previously described10 using pan CD45-APC, CD31-APC, Sca-1 (Ly-6A/E)-APC-Cy7 (PharMingen), EpCAM-PE-Cy7 (BioLegend) with 4′, 6-diamidino-2-phenylindole (DAPI) (Sigma) staining to eliminate dead cells. Cell sorting was performed with a BD FACS Aria and data were analyzed with FlowJo software (Tree Star, Inc.). Amplified MEC or MSC were used for 3D co-cultures of Epcam+ Sca-1− cells and Epcam+ Sca-1+ cells or dissociated organoids from the latter. MEC or MSC were added to growth factor–reduced Matrigel (BD Bioscience) at 1 × 106 cells/ml. Sorted Epcam+ Sca-1+ and Epcam+ Sca-1− cells were resuspended in the MEC or MSC containing Matrigel, which was prediluted at a ratio of 1:1 with medium and added to a 24-well transwell filter inserts with 0.4um pore (Corning) in a 24-well tissue culture plate containing 500 μl of medium. Dulbecco’s Modified Eagle’s Medium/F12 (Invitrogen) was supplemented with 10% FBS, penicillin/streptomycin, 1 mM HEPES, and insulin/transferrin/selenium (Sigma) for all cultures. Cultures were incubated at 37 °C in a humidified incubator (5% CO2), and the medium was replaced every other day for up to 14 days. For serial passages, Epcam+ Sca-1+ and Epcam+Sca-1− cells 3D cocultures were dissociated in dispase (BD Bioscience) and trypsin (GIBCO) to generate a single-cell suspension followed by FACS for GFP (or DsRED). GFP+ (or DsRED+) cells were resuspended with fresh MEC or MSC/Matrigel mixtures. Typically, 2,000 cells were plated for primary organoid (1°) formation. Primary organoids were dissociated, FACS sorted for GFP (or DsRED), and 2,500 GFP+ (or DsRED+) cells were replated with fresh MEC or MSC/Matrigel mixture for secondary (2°) and subsequent (3° and 4°) organoid formation biweekly.

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Manufacturer protocol

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