Primary ASMCs culture from mice

Primary ASMCs were isolated by a modified protocol according to a previous study (18). We entirely separated the trachea and lungs, carefully removed the extraneous membranes and connective tissues, and cut the tissue into cross-sections of 2–3 mm. The tissue was placed in a culture flask and immersed in Dulbecco’s modified Eagle medium (DMEM) containing 20% fetal bovine serum, 4 mM glutamine, and 1% penicillin-streptomycin. The cultured specimens were incubated in a 5% CO₂ incubator at 37 °C until 80% confluent. We obtained the desired cells which could be used for experiments after purification.