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. 2023 May 30;13:8743. doi: 10.1038/s41598-023-35070-2

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Generation and characterisation of hACE2 receptor stably expressing HEK-293T cell line. The VSV-G lentiviral particles were produced with pLenti-hACE2-P2A-PuroR plasmid and transduced into HEK-293T cells. The stable expressing hACE2 HEK-293T cell line was selected with puromycin. The expression level of hACE2 in 293T-hACE2 cells were analysed by qPCR (A) and western blotting (with 20 µg of protein lysates) techniques (B). The functional characterization of 293T-hACE2 stable cells were done by RBD-Biotin surface staining. Graphical representation of RBD-biotin surface staining principle (C). After RBD-Biotin surface staining, the ancestral SARS-CoV-2 RBD binding hACE2 level on surface of 293T-hACE2 cells were quantified by flow cytometry (D, E) (MFI-mean florescent intensity) and RBD-hACE2 interaction was visualised by confocal microscopy (F).