Gibco™ DMEM, high glucose

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	PAECs and the explant pieces of smooth muscle were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 U/ml), 5 mM L-glutamine and 1.5 g/l sodium bicarbonate. The medium was changed every 2 days. The cells were cultured in an incubator at 37ºC and maintained in a humidified atmosphere with 5% CO2.

Protocol tips
PAECs and the explant pieces of smooth muscle were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 U/ml), 5 mM L-glutamine and 1.5 g/l sodium bicarbonate. The medium was changed every 2 days. The cells were cultured in an incubator at 37ºC and maintained in a humidified atmosphere with 5% CO2.

Publication protocol

PAECs and PASMCs were cultured from porcine pulmonary arteries as previously described (18,21). PAECs and PASMCs, which were used between passages 2 and 6, were isolated from porcine pulmonary arteries obtained from a local abattoir within 1 h of slaughter. Endothelial cells were digested with 1.7 mg/ml collagenase II, then the pulmonary arteries were cut open along the longitudinal axis, and the residual endothelium was gently removed by scraping the luminal surface and washed away with phosphate-buffered saline (PBS). The adventitia layer was removed by blunt dissection, and the medial smooth muscle tissue was minced into 1 mm3 explant pieces. PAECs and the explant pieces of smooth muscle were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 U/ml), 5 mM L-glutamine and 1.5 g/l sodium bicarbonate. The medium was changed every 2 days. The cells were cultured in an incubator at 37ºC and maintained in a humidified atmosphere with 5% CO2. The purity and identity of the endothelial cells and smooth muscle cells were verified by their typical morphological patterns and by immunofluorescent staining for factor VIII-related antigen and α-smooth muscle actin (α-SMA), respectively (16,21). All passages were performed using 0.05% trypsin and 0.02% EDTA. Cell viability was assessed using the CCK-8 assay.

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