2.5. Cell Culture

The cell line NCI-H441 (ATCC^® HTB174™) was a kind gift from Prof. Dr. Mike Althaus (Bonn-Rhein-Sieg University of Applied Sciences, Rheinbach, Germany). NCI-H441 cells were cultured in uncoated T75 flasks in RPMI 1640 medium with supplements (1% antibiotic/antimycotic solution, 1% sodium pyruvate, 1% ITS media supplement, and 10% fetal bovine serum) at 37 °C, 5% CO₂ and 95% rH. For Ussing chamber experiments, cells were seeded onto uncoated Costar Transwell^®permeable filters (Ø = 6.5 mm; REF 3470; Corning Inc., Lowell, MA, USA) with a density of 1 × 10⁶ cells/cm². One day after seeding, the NCI-H441 medium was replaced by NCI-H441 medium supplemented with 200 nM dexamethasone. For cultivation of the cells under ALI conditions, the medium on the apical side of the filters was removed one day after seeding and cells were only supplied with the medium on the basolateral side. The filters were cultured for at least 7 days at 37 °C, 5% CO₂ and 95% rH before experiments were conducted. For transfection experiments with NCI-H441 cells grown on filters, the medium was replaced by antibiotic-free NCI-H441 medium without dexamethasone 24 h before transfection. For fluorescence optical experiments, cells were seeded on glass cover slips (Ø = 12 mm; Carl Roth GmbH & Co. KG, Karlsruhe, Germany) with a density of 1 × 10⁵ one day before transfection and incubated in antibiotic-free medium.