Human pulmonary artery endothelial cells (HPAECs) and EGM-2 growth media kit were obtained from Lonza (Allendale, NJ) and cultured according to the manufacturer’s instructions. For the coculture experiments, HPAEC were used at early passages. Before the experiments, the EGM media was changed to basal media supplemented with 2% fetal bovine serum (FBS), unless otherwise specified.
TF-1 cells were cultured as described previously.9 Briefly, TF-1 cells, an erythroleukemia cell line, were obtained from ATCC (Manassas, VA) and genetically engineered to overexpress GPA. These cells were graciously donated to us by the Kingsbury Laboratory at the University of Maryland School of Medicine. Cells were cultured in RPMI medium (Gibco, ThermoFisher, Waltham, MA) containing 10% FBS and 1 ng/mL GM-CSF (Biolegend, San Diego, CA). Cells were split every 48 to 72 hours and plated at a density of 1 million cells per mL of medium. GPA overexpression was confirmed after fluorescent antibody staining and flow cytometry analysis, as described above, for RBC surface GPA.
TF-1 cells were cultured as described previously.9 Briefly, TF-1 cells, an erythroleukemia cell line, were obtained from ATCC (Manassas, VA) and genetically engineered to overexpress GPA. These cells were graciously donated to us by the Kingsbury Laboratory at the University of Maryland School of Medicine. Cells were cultured in RPMI medium (Gibco, ThermoFisher, Waltham, MA) containing 10% FBS and 1 ng/mL GM-CSF (Biolegend, San Diego, CA). Cells were split every 48 to 72 hours and plated at a density of 1 million cells per mL of medium. GPA overexpression was confirmed after fluorescent antibody staining and flow cytometry analysis, as described above, for RBC surface GPA.