Transfection and Analysis of SARS-CoV-2 Spike Protein

HEK293T cells were transiently transfected with mRNA encoding SARS-CoV-2 WT S or S-2P protein using a TranIT mRNA transfection kit (Mirus). After 24 hr, the cells were harvested and resuspended in FACS buffer (1X PBS, 3% FBS, 0.05% sodium azide). To detect surface protein expression, the cells were stained with 10 μg/mL ACE2-FLAG (Sigma) or 10 μg/mL CR3022^{35 (link)} in FACS buffer for 30 min on ice. Thereafter, cells were washed twice in FACS buffer and incubated with FITC anti-FLAG (Sigma) or Alexafluor 647 goat anti-human IgG (Southern Biotech) in FACS buffer for 30 min on ice. Live/Dead aqua fixable stain (Invitrogen) were utilized to assess viability. Data acquisition was performed on a BD LSRII Fortessa instrument (BD Biosciences) and analyzed by FlowJo software v10 (Tree Star, Inc.)

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Corbett K.S., Edwards D.K., Leist S.R., Abiona O.M., Boyoglu-Barnum S., Gillespie R.A., Himansu S., Schäfer A., Ziwawo C.T., DiPiazza A.T., Dinnon K.H., Elbashir S.M., Shaw C.A., Woods A., Fritch E.J., Martinez D.R., Bock K.W., Minai M., Nagata B.M., Hutchinson G.B., Wu K., Henry C., Bahi K., Garcia-Dominguez D., Ma L., Renzi I., Kong W.P., Schmidt S.D., Wang L., Zhang Y., Phung E., Chang L.A., Loomis R.J., Altaras N.E., Narayanan E., Metkar M., Presnyak V., Liu C., Louder M.K., Shi W., Leung K., Yang E.S., West A., Gully K.L., Stevens L.J., Wang N., Wrapp D., Doria-Rose N.A., Stewart-Jones G., Bennett H., Alvarado G.S., Nason M.C., Ruckwardt T.J., McLellan J.S., Denison M.R., Chappell J.D., Moore I.N., Morabito K.M., Mascola J.R., Baric R.S., Carfi A, & Graham B.S. (2020). SARS-CoV-2 mRNA Vaccine Design Enabled by Prototype Pathogen Preparedness. Nature, 586(7830), 567-571.

Publication 2020

TranIT mRNA transfection kit ACE2-FLAG FITC anti-FLAG Alexafluor 647 goat anti-human IgG Live/Dead aqua fixable stain BD LSRII Fortessa instrument

Ace2 Alexafluor 647 Anti igg Buffer Cells Fitc Goat Human Mrna Protein Sars cov 2 Sodium azide Stain Surface protein Transfection Tree

Corresponding Organization :

Other organizations : National Institute of Allergy and Infectious Diseases, National Institutes of Health, Moderna Therapeutics (United States), University of North Carolina at Chapel Hill, George Washington University, Vanderbilt University Medical Center, The University of Texas at Austin

Top 5 similar protocols

Variable analysis

independent variables

Transfection of HEK293T cells with mRNA encoding SARS-CoV-2 WT S or S-2P protein

dependent variables

Surface protein expression of SARS-CoV-2 S or S-2P protein on HEK293T cells
Viability of HEK293T cells

control variables

Cell line (HEK293T)
Transfection reagent (TranIT mRNA transfection kit)
Staining conditions (10 μg/mL ACE2-FLAG or 10 μg/mL CR3022, 30 min on ice)
Washing procedure (twice in FACS buffer)
Secondary staining (FITC anti-FLAG or Alexa Fluor 647 goat anti-human IgG, 30 min on ice)
Viability staining (Live/Dead aqua fixable stain)
Flow cytometry (BD LSRII Fortessa instrument)

positive controls

Cells transfected with mRNA encoding SARS-CoV-2 WT S protein
Cells transfected with mRNA encoding SARS-CoV-2 S-2P protein

negative controls

Untransfected HEK293T cells

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