Vero (ATCC CCL-81) cells and Vero-furin cells (Mukherjee et al., 2016) were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and 100 U/ml of penicillin–streptomycin Spike ectodomain, human mAbs and Fabs were expressed in HEK293T cells cultured in FreeStyle 293 Expression Medium (12338018, ThermoFisher) at 37°C with 8% CO2. Nucleoprotein was expressed using 2-L cultures of Rosettagami2(DE3)pLysS bacteria (Novagen) in terrific broth medium containing 40 mg/L kanamycin, at 15°C for 40 hr following induction with Isopropyl β-D-1-thiogalactopyranoside (1mM final concentration, Meridian Bioscience). For ACE2 and RBD, transient expression used Expi293F cells (Thermo Fisher, Cat# A14527) grown in Expi293 Expression Medium (Thermo Fisher Cat# A1435103) in suspension with 8% CO2 at 30 or 37°C and shaking at 130 rpm. For production of Spike protein for structural analysis, HEKExpi293F cells (Thermo Fisher Scientific) were transfected with the construct together with a phiC31 integrase expression plasmid and grown in adhesion roller bottles with the high glucose DMEM (Sigma) with 2% FBS for 6 days at 30°C. His-tagged RBD for structural analysis was expressed in a stable HEK293S cell line cultured in DMEM (high glucose, Sigma) supplemented with 10% FBS (Invitrogen), 1 mM glutamine and 1x non-essential amino acids at 37 °C. Cells were transferred to roller bottles (Greiner) and cultured in DMEM supplemented with 2% FBS, 1 mM glutamine and 1x non-essential amino acids at 30 °C for 10 days for protein expression. For plaque assays Vero-furin cells (Mukherjee et al.,2016) were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES, and 100 U/ml of penicillin–streptomycin.

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