Endothelial cell growth medium 2 egm 2tm supplemental bullet kit

HEK293T cells used for lentivirus production were obtained from ATCC and maintained in DMEM, high glucose, and GlutaMAX^TM (Gibco) supplemented with 10% FBS (Corning) and 1% penicillin/streptomycin (Gibco) (D10 medium) at 37 °C with 5% CO₂. Calu-3 human lung epithelial cells were obtained from the UC Berkeley Cell Culture Facility and maintained in D10 medium at 37 °C with 5% CO₂. Human pulmonary microvascular endothelial cells (HPMEC) [line HpMEC-ST1.6 R] were a gift from Dr. J.C. Kirkpatrick at Johannes Gutenberg University, Germany. These cells were isolated from an adult human male donor, and an immortalized clone was selected that displayed all major phenotypic makers of pulmonary endothelial cells^{77 (link)}. HPMECs were cultured in endothelial cell growth basal medium 2 supplemented with an Endothelial Cell Growth Medium-2 (EGM-2^TM) supplemental bullet kit (Lonza). Cells were maintained at 37 °C with 5% CO₂. This study produced several genetically modified versions of these parental cells, including HPMEC overexpressing the human ACE2 (hACE2) gene (HPMEC/ACE2) as well several knockout cell lines. The hACE2 encoding plasmid was a gift from Hyeryun Choe (Addgene plasmid #1786; http://n2t.net/addgene:1786; RRID:Addgene_1786)^{70 (link)}. Vero-E6 cells were used for SARS-CoV-2 titration and maintained in D10 media at 37 °C with 5% CO₂.