*4.1. Cell Culture*

Primary rat alveolar epithelial type II (ATII) cells were isolated as previously described [14]. Isolation of ATII cells from rats was conducted according to the legal regulations of the German Animal Welfare Act and was approved by the regional authority (Regierungspräsidium Gießen, reference number A12/2013 from March 28, 2013 and reference number G41/2018 from July 17, 2018) of the State of Hessen, Germany. ATII, human alveolar epithelial A549 (ATCC, CCL 185), and A549 cells stably expressing Na,K-ATPase α1-subunit fused to a green fluorescent protein (A549-α1-GFP) were grown in Dulbecco's modified Eagle's media (DMEM, Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories, Egelsbach, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin, as previously described [14]. For experiments, 600,000 A549 or 2,000,000 ATII cells were grown on 60-mm culture dishes (Sarstedt, Nümbrecht, Germany) in 4 mL of culture media. Alternatively, 200,000 A549 or 1,500,000 ATII cells were seeded on permeable membrane supports (BD Falcon, Heidelberg, Germany) with maintaining a liquid/liquid interface. Subconfluent cell monolayers were used for experiments. Cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 ◦C.
