Dulbecco s modified eagle medium f12

Human club cell line H441 (ATCC HTB‐174) was expanded as submerged monolayers in Roswell Park Memorial Institute‐1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) heat‐inactivated fetal bovine serum and 0.5% (v/v) penicillin/streptomycin (P/S). To act as epithelial barriers in neutrophil transmigration experiments, H441 cells were cultured at ALI as previously described.⁷ H441 cells were seeded onto porous Alvetex 3D Scaffolds (REPROCELL, Beltsville, MD, USA), coated with gelled collagen, at a density of 2.5 × 10⁵ cells per scaffold. The larger void size of scaffolds (33–55 µm) compared with pores on a Transwell membrane (0.4 µm) has been shown to facilitate neutrophil migration and swarming behavior.⁷ Scaffolds were initially grown submerged in Dulbecco’s Modified Eagle Medium/F12 (Corning) supplemented with 10% (v/v) heat‐inactivated fetal bovine serum and 0.5% (v/v) P/S for 2 days. Apical media were then removed and scaffolds were cultured at ALI for 2 weeks, fed basolaterally with Dulbecco’s Modified Eagle Medium/F12 (Corning) supplemented with 2% (v/v) Ultroser G (Pall Life Sciences, Port Washington, NY, USA) and 0.5% (v/v) P/S with media changes every 48 h.

FACS-sorted mouse club cells (5 × 10³) or AT2 cells (2 × 10⁴) were co-cultured with MLg (2 × 10⁵), mouse lung fibroblast cells (ATCC, Rockefeller, MD, USA),
in Matrigel/culture medium (1:1) respectively. The culture medium included Dulbecco’s modified Eagle Medium/F12 (Corning, Corning, NY, USA), 1% insulin/transferrin/selenium (Sigma), 10% FBS,
100 IU/ml penicillin, 100 µg/ml streptomycin, and 10 µM SB431542 (Sigma). In the treatment group, 10 µM HCQ was added to the culture
medium. Then, the mixture was transferred to the Transwell inserts in 24-well plates (Corning) containing the culture medium at the bottom. An incubator with a humidified 37°C with 5%
CO₂ was used to maintain cultures, and the culture medium was changed every other day. The organoids were imaged using an inverted fluorescence microscope (Olympus, Tokyo,
Japan). The colony-forming efficiency (CFE) was calculated as the number of organoids (greater than 50 µm in diameter) in each insert as a percentage of seeded epithelial
cells.