Nucleopore polycarbonate track etch membrane

Lung explants were cultured ex vivo following established protocols^{37 (link)}. Lungs were dissected in cold PBS supplemented with antibiotics (50 units/ml of penicillin and streptomycin) and cultured on porous membranes (nucleopore polycarbonate track-etch membrane, 8 μm pore size, 25 mm diameter; Whatman) floating on DMEM/F12 medium (without HEPES) supplemented with 5% fetal bovine serum (FBS, heat inactivated; Atlanta Biologicals) and antibiotics (50 units/ml of penicillin and streptomycin) within a glass-bottom dish. Lungs were cultured within a stage-top incubator (Pathology Devices) on an inverted microscope (Nikon Ti) and imaged under brightfield (1–2 ms exposure). Every hour for 12 hours, a five-minute timelapse was taken with frames acquired every three seconds. Timelapses from hours 6 to 12 were analyzed for the presence of spontaneous smooth muscle contractions, visible by deformations of the epithelium, and compared using a two-sided t-test in GraphPad Prism. For EdU incorporation experiments, we first cultured E12.5 lungs on porous membranes for 1 hour and then added EdU to the culture medium for 15 minutes prior to fixation and subsequent reactions using the Click-iT EdU Alexa Fluor 488 Kit (Thermo Fisher Scientific).

Lungs were dissected in PBS and cultured on porous membranes (nucleopore polycarbonate track-etch membrane, 8 μm pore size, 25 mm diameter; Whatman) in DMEM/F12 medium (without HEPES) supplemented with 5% fetal bovine serum (FBS, heat inactivated; Atlanta Biologicals) and antibiotics (50 units/ml of penicillin and streptomycin). E11.5 lungs were isolated from iDTR^fl/fl females bred to Tagln-Cre males, and then treated with 0.2 ng/μl DT (Sigma-Aldrich). Before adding it to the culture medium, DT was unnicked by incubating with trypsin at a ratio of 1:10,000 in PBS for 15 min at 37°C. iDTR^fl/+ littermates were used as controls.