Culturing Murine Pulmonary Epithelial Cells

The mouse pulmonary epithelial cell lines MLE-12 and A549 were obtained from ATCC (VA, USA) and cultured in DMEM containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin in a 5% CO₂ atmosphere at 37°C. MLE-12 or A549 cells were seeded in 6-well plates or 24-well plates overnight and then treated with HDM extract or the cytokine IL-33 for the indicated durations. Primary alveolar epithelial cells from mice were purified using 0.1% collagenase, 0.25% trypsin, and DNase I and were selected with mouse IgG (36111ES60, Yeasen, China) as previously described (27 (link)). To exclude the potential effects of lipopolysaccharide (LPS) contamination, HDM extract was treated with the ToxinEraser^TM endotoxin removal Kit (L00338, Genscript, China). The purified product was the major constituent of HDM.