Human ACE2 293T Cell Line

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Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase

Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding luciferase. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein (full length or truncated forms) and encoding the firefly luciferase protein. 10 μl of concentrated virus (21X) from each prep was used to transduce the Human ACE2 293T Cell Line. Luciferase activity was measured 6 days post-transduction using a control virus lacking an envelope protein to provide a background signal for analysis of the luciferase activity.

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Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding ZsGreen1

Transduction of Human ACE2 293T Cell Line with SARS-CoV-2 pseudoviruses encoding ZsGreen1. Lenti-X SARS-CoV-2 Packaging Single Shots were used to generate lentiviral particles pseudotyped with either WT or D614G variants of the spike protein (truncated form) and encoding the fluorescent protein ZsGreen1. 100 μl of supernatant from each prep was used to transduce the Human ACE2 293T Cell Line. Panel A. Microscopy images of transduced cells at 72 hours post-infection. Panel B. Transduction efficiencies for each sample were measured by flow cytometry 6 days post-transduction.

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Analysis of ACE2 expression by flow cytometry

Analysis of ACE2 expression by flow cytometry. ACE2 expression at the cell surface of the Human ACE2 293T Cell Line was detected by flow cytometry using an Alexa Fluor 488-conjugated monoclonal anti-ACE2 antibody (R&D Systems, Cat. # FAB9332G). Non-labelled cells were used as a negative control.

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Confirmation of stable ACE2 expression across multiple cell passages

Confirmation of stable ACE2 expression across multiple cell passages. Relative mRNA expression levels of ACE2 across successive passages of the Human ACE2 293T Cell Line were determined by RT-qPCR using the One Step PrimeScript RT-PCR Kit (Perfect Real Time) (Cat. # RR064A). Ct values for ACE2 expression were normalized to Ct values for the housekeeping gene ACTB in triplicates. ACE2 expression in the stable cell line was determined relative to a 293T cell line by calculating 2^(–ΔΔCt).

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Neutralization data with convalescent serum

Use of the Human ACE2 293T Cell Line in neutralization assays employing pooled non-human primate convalescent serum to SARS-CoV-2 as a blocking agent. WT (truncated) SARS-CoV-2 pseudovirus was incubated with serial dilutions of the convalescent serum (BEI Resources, Cat. # NR-52401) and applied to the Human ACE2 293T Cell Line. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate. Negative control was performed using serum from healthy macaques.

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Neutralization data with ACE2 protein as a blocking agent

Use of the Human ACE2 293T Cell Line in neutralization assays employing soluble ACE2 protein as a blocking agent. Panels A and B. Serial dilutions of soluble ACE2 protein fused to the Fc domain from IgG (ACE2-Fc) were applied, along with SARS-CoV-2 pseudovirus, to the Human ACE2 293T Cell Line. Luciferase levels were measured 3 days post-infection as a readout for virus infectivity. Data are graphed as percent neutralization relative to virus-only control infection. Values are mean ±SD and experiments were performed in triplicate.

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631289: Human ACE2 293T Cell Line