NCI-H441 Cell Culture for Ussing Assay

Human distal lung epithelial cell line NCI-H441 was obtained from the American Type Culture Collection (ATCC) and cultured as previously described [16 (link)]. H441 cells were grown in RPMI medium (ATCC) containing 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). For Ussing chamber assays, cells were seeded on permeable support filters (Costar) at a supraconfluent density (∼5 × 10⁶ cells/cm²) and incubated in a humidified atmosphere of 5% CO₂ at 37°C. Dexamethasone (250 nM, Sigma) was supplemented to stimulate ENaC expression. Cells reached confluency in the Costar Transwells 24 h after plating. At this point, media and nonadherent cells in the apical compartment were removed to adapt the cells to the air-liquid interface culture. Culture media in the basolateral compartment were replaced every other day, whereas the apical surface was rinsed with PBS. Transepithelial resistance was measured by an epithelial tissue volt-ohm-meter (World Precision Instruments). Highly polarized tight monolayers with resistance >500 Ω cm² were used for measuring short-circuit current (I_sc) levels of transepithelium.