For recombinant proteins with the Fc domain, 293T cells were transfected with a vector containing human ACE2 (10 μg pCAG-hACE2/T75). At 48 h post-transfection, the cells were washed with PBS 3 times. The cells were harvested and seeded into an Eppendorf tube (1 × 106/mL). An RBD-His protein (1 µg/mL) was added to the cells in the presence or absence of mice sera at indicated dilution (1:10). After incubation for 30 min at room temperature (RT), cells were rinsed with PBS. We then added the anti-6X His tag antibody-FITC (Abcam, ab1206, Cambridge, UK) at 1:100 dilution, and incubated at 4 °C for 30 min. For the positive control, RBD-His and anti-6X His tag antibody-FITC without antisera were added; for the negative control, neither pooled mice serum nor RBD-His was added. After thorough washing, the cells were resuspended in 1 mL PBS and fixed with 1% paraformaldehyde. The inhibitory effect was analyzed by flow cytometry (BD Biosciences, San Diego, CA, USA) and analyzed by FlowJo v10 software (FlowJo, LLC, Ashland, OR, USA).
For His-tag recombinant proteins, RBD-Fc followed by an anti-human IgG (Fc specific)-FITC conjugate (Sigma, 079M4801V, St. Louis, MO, USA) was used for analysis.
For His-tag recombinant proteins, RBD-Fc followed by an anti-human IgG (Fc specific)-FITC conjugate (Sigma, 079M4801V, St. Louis, MO, USA) was used for analysis.