Percoll

Buffy coats from healthy donors were obtained after written informed consent (Sanquin Blood Bank, Nijmegen, The Netherlands). Ethical approval was obtained from the CMO Arnhem-Nijmegen (NL32 357.091.10). Isolation was performed by differential density centrifugation over Ficoll-Paque (GE Healthcare, Chalfont St Giles, UK). Subsequently, isolation of monocytes was performed with a hyper-osmotic Percoll (Sigma-Aldrich, St Louis, MO, USA) density gradient centrifugation and washed once with pyrogen-free cold phosphate buffered saline (PBS). Cells were resuspended and later cultured in RPMI 1640 Dutch modified medium (Invitrogen, Waltham, MA, USA) supplemented with 5 μg/mL gentamicin (Centraform, Etten-Leur, the Netherlands), 2 mM Glutamax (Gibco, Walthan, MA, USA), and 1 mM pyruvate (Gibco). To ensure maximal purity, Percoll-isolated monocytes were left to adhere to polystyrene flat bottom plates (Corning, Sigma-Aldrish, New York, NY, USA) for 1 h at 37 °C 5% CO₂ and then washed once with warm PBS.

Epithelial cells were isolated from mouse lung tissue by mechanical separation using a sterile sieve followed by discontinuous density gradient centrifugation using a Percoll (GE Healthcare Life Sciences, Chicago, IL, USA)⁶⁸ . Three Percoll solutions (25%, 40%, and 75%) were prepared, and cells from lung tissue were resuspended in the 40% solution, and the three different gradients were carefully placed in a 15 ml centrifuge tube (Corning, USA) in the order 25% − 40% − 75% from top to bottom. Cells were spun at 780 × g for 20 min, with minimal acceleration and deceleration set. Epithelial cells were placed on the 25% and 40% Percoll solutions interface and then aspirated and washed several times with DMEM containing 10% FBS. Approximately 2 × 10⁶ cells were inoculated in a 6-well plate coated with collagen.

Epithelial cells were isolated from mouse lung tissues by using a mechanical dissociation with sterile sieve meshes followed by a discontinuous density-gradient centrifugation using Percoll (GE Healthcare Life Sciences, Chicago, IL, USA) in accordance with previous studies [17 (link),18 (link)] with some modifications. Three Percoll solutions (25%, 40%, and 75%) were prepared, the cells from lung tissues were resuspended in 40% solution, and three distinct gradients were carefully placed in the order of 25%–40%–75% from top to bottom in a 15 mL centrifuge tube (Corning, Glendale, AZ, USA). The cells were spun at 780× g for 20 min in a centrifuge (AX-511) (Tomy, Tokyo, Japan) at the setting of minimal acceleration and deceleration. The epithelial cells were centered at an interface between 25% and 40% Percoll solution and were then taken and washed extensively with RPMI1640 (Nacalai) containing 10% FBS (Equitech-Bio) and penicillin/streptomycin (Nacalai).