Htepc

For calcium imaging, we used the human tracheal epithelial cell line (HTEpC, C12644, PromoCell, Heidelberg, Germany). Cells were cultivated in an Airway Epithelial Cell Growth Medium Kit (C-21160) containing the Airway Epithelial Cell Growth Medium Supplement Pack (C-39160, PromoCell, Heidelberg, Germany). The airway epithelial cells were kept in a humidified chamber at 37 °C with air containing 5% CO₂. For the Ca²⁺ imaging, cells were seeded onto laminin-coated coverslips. Dye loading with 2.5 µM FURA-2 AM (Biotium, Fremont, CA, USA) in darkness was performed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer for 30 min at 37 °C. For the contents of the HEPES buffer, see the section on drugs and buffer solutions. After the loading period, the cells were rinsed in fresh HEPES buffer and were transferred to the recording chamber of an upright fluorescence microscope equipped with 20 × immersion lenses (BX50 WI, Olympus, Hamburg, Germany), which contains 2 ml HEPES. The excitation light was provided by a 50 W xenon lamp. The microscope was equipped with a dichromatic excitation longpass mirror (400 nm).
The ratiometric dye, FURA-2 AM, was excited at 340 nm and 380 nm (± 8 nm) when equipped with bandpass excitation filters. The emitted fluorescence was directed through a dichromatic shortpass filter of 560 nm to a bandpass filter of 510 nm.