Freestyle 293 expression media

Expression
of the subtype A 92UG037.8 gp140 trimer was previously described.^{8 (link),14 (link)} Briefly, a stably transfected 293T cell line was expanded to confluence
in DMEM supplemented with 10% FBS and subsequently media exchanged
to Freestyle 293 expression media (Invitrogen). Cell supernatants
were collected 96 h after media exchange and subjected to standard
Ni-NTA (Qiagen) affinity chromatography followed by Superose 6 (GE
Healthcare) size exclusion chromatography in 25 mM TRIS (pH 7.5) plus
150 mM NaCl. Fractions containing the purified protein were subjected
to SDS-PAGE electrophoresis in order to monitor purity before fractions
were pooled, concentrated, and flash-frozen in liquid nitrogen and
stored at −80 °C.

FreeStyle 293-F
suspension cells were cultured in FreeStyle 293 Expression
Media (Invitrogen) at 37 °C in a humidified shaking platform
incubator (Kuhner) with 5% CO₂. For transfection, cells
were pelleted at 500g and resuspended in fresh media.
For small-scale (1 mL of cells at 1 × 10⁶/mL) transient
transfections performed in 24-well nontreated tissue culture plates,
2 μg of polyethylenimine (PEI) was added to 0.5 μg of
diluted plasmid DNA in a final volume of 100 μL. For small-scale
(.25 mL of cells at 1 × 10⁶/mL) transient transfections
performed in 48-well nontreated tissue culture plates, .5 μg
of polyethylenimine (PEI) was added to 0.125 μg of diluted plasmid
DNA in a final volume of 25 μL. DNA–PEI complexes were
incubated at room temperature for 10 min and then added directly to
cells in 48-well plates. FreeStyle 293-F cells were utilized to screen
for spike protein interactions, so genes that were transfected into
FreeStyle 293-F cells included human ACE2 and CD26, mouse ACE2, the
secretome library, and GFP controls.

FreeStyle293F cells (ThermoFisher Scientific) and HEK293-S cells (ThermoFisher Scientific) were cultured in FreeStyle293 expression media (Life Technologies), cultured at 37°C with 8% CO₂ while shaking at 130 rpm. HEK293-T cells (ATCC) and Vero E6 cells (ATCC) were cultured at 37°C in the presence of 5% CO₂ in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin, 1% streptomycin, 2 mM l-glutamine, non-essential amino acids (Invitrogen) and 1 mM sodium pyruvate. Huh7.5 cells (provided by Dr. Deborah R. Taylor) were cultured at 37°C with 8% CO₂ in flasks with DMEM + 10% FBS. ExpiCHO-S cells (GIBCO) were cultured at 37°C with 8% CO₂ while shaking at 130 rpm in ExpiCHO expression media (GIBCO). Cells lines were not tested for mycoplasma contamination nor authenticated.