Immunofluorescence staining
This was performed to detect the binding between SARS-CoV-2 RBD and hACE2 receptor in hACE2/293T stable cell lines.33 SARS-CoV and MERS-CoV RBDs were used as controls. Briefly, cells were sequentially incubated with Fc-fused SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (20 μg/ml) and hACE2-specific goat antibody (5 μg/ml) for 30 min at room temperature. After three washes, the cells were incubated with FITC-labeled goat anti-human IgG (Fc) antibody (1:500; Thermo Fisher Scientific), or Alexa-Fluor 647-labeled anti-goat antibody (1:200 dilution; Abcam) for 30 min at room temperature. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) for 5 min and mounted in VectaMount Permanent Mounting Medium. The samples were imaged on a confocal microscope (Zeiss LSM 880), and the images were prepared using the ZEN software.