Protocol detail

SARS-CoV-2 Protein Expression in HeLa Cells

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HeLa cells were transiently transfected with pLVX-EF1alpha-nCoV2019 2xStrep-tagged SARS-CoV2 ORF constructs [47 (link)] using TransIT-HeLa Monster (Mirus Bio). After 48 hours transfection, cells were detached and the cell population equally split. Half of the cells were permeabilised and intracellular Strep levels were measured by flow cytometry. Surface tetherin staining was performed on the remaining half of the cells.

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Stewart H., Palmulli R., Johansen K.H., McGovern N., Shehata O.M., Carnell G.W., Jackson H.K., Lee J.S., Brown J.C., Burgoyne T., Heeney J.L., Okkenhaug K., Firth A.E., Peden A.A, & Edgar J.R. (2022). Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic. bioRxiv.

Publication Preprint 2022

TransIT-HeLa Monster

Cells Flow cytometry Hela Intracellular Sars Strep Tetherin Transfection

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Variable analysis

independent variables

Transfection of HeLa cells with pLVX-EF1alpha-nCoV2019 2xStrep-tagged SARS-CoV2 ORF constructs

dependent variables

Intracellular Strep levels measured by flow cytometry
Surface tetherin staining

control variables

Mirus Bio TransIT-HeLa Monster used for transfection
Cell population equally split after 48 hours of transfection
Half of the cells permeabilised for intracellular Strep level measurement
Remaining half of the cells used for surface tetherin staining

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