2.2. Isolation of Mouse Pleural Mesothelial Cells (PMCs)

Primary mouse PMCs were isolated from the diaphragm, the external surface of the heart, and lungs of mice 3–4 weeks old as previously described [13]. Pieces of diaphragm and adjunct organs were placed in a 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solution for 1 h. After removing the intact tissues and debris, the cell suspension was centrifuged at 1500 rotations per minute for 5 min. The pellet was resuspended and maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 15% fetal bovine serum (FBS; Corning costar, Corning, NY, USA) and 1% penicillin/streptomycin (Sigma, St Loise, MO, USA) in a humidified incubator with 5% CO₂ at 37 °C. The following day, the cells were washed three times with phosphate buffered saline (PBS) to remove non-adherent cells and left until they were confluent (media change every 2 days). When a homogeneous population of cobblestone PMCs was shown at passage ~3–4, the cells were subjected to experiments described below.