Cell Culture Conditions for SARS-CoV-2 Research

Vero E6 cells (ATCC) were cultured at 30°C in the presence of 5% CO₂ in medium composed of high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 5% (v/v) heat-inactivated fetal bovine serum (FBS) (VWR). TMPRSS expressing Vero E6 cells (gift from Rhea Coler) were cultured in DMEM supplemented with 5% (v/v) FBS and 1 mg/ml geneticin. 293T/17 and 293T-hACE2 (Cawford et al., 2020 (link)) cells (Life Technologies; Cat# R70007; RRID:CVCL_6911) were cultured in DMEM (Gibco) supplemented with 10% FBS, penicillin/streptomycin, 10 mM HEPES, and with 0.1 mM MEM non-essential amino acids (Thermo Fisher). All experiments were performed with cells passaged less than 15 times. The identities of cell lines were confirmed by chromosomal marker analysis and tested negative for mycoplasma using a MycoStrip (InvivoGen).

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Mast F.D., Fridy P.C., Ketaren N.E., Wang J., Jacobs E.Y., Olivier J.P., Sanyal T., Molloy K.R., Schmidt F., Rutkowska M., Weisblum Y., Rich L.M., Vanderwall E.R., Dambrauskas N., Vigdorovich V., Keegan S., Jiler J.B., Stein M.E., Olinares P.D., Herlands L., Hatziioannou T., Sather D.N., Debley J.S., Fenyö D., Sali A., Bieniasz P.D., Aitchison J.D., Chait B.T, & Rout M.P. (2021). Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape. eLife, 10, e73027.

Publication 2021

Vero E6 cells DMEM MEM non-essential amino acids MycoStrip

Cell lines Cells Chromosomal marker Eagle Essential amino acids Fetal bovine serum Geneticin Glucose Hepes Mycoplasma Penicillin Rhea Streptomycin Vero cells

Corresponding Organization :

Other organizations : Infectious Disease Research Institute, Rockefeller University, St. John's School, University of California, San Francisco, QB3, Institute for Systems Biology, University of Washington, Seattle Children's Hospital, Howard Hughes Medical Institute

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Temperature (30°C)
Presence of 5% CO2

dependent variables

Cell growth and/or proliferation
Protein expression and/or viral infection

control variables

Cell lines (Vero E6, TMPRSS expressing Vero E6, 293T/17, 293T-hACE2)
Culture medium (high-glucose DMEM, 5% FBS, geneticin for TMPRSS expressing Vero E6 cells)
Cell passage number (less than 15 passages)

positive controls

TMPRSS expressing Vero E6 cells (gift from Rhea Coler)

negative controls

Vero E6 cells without TMPRSS expression
293T/17 cells without hACE2 expression

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