2.6. Cell-based Spike-ACE2 inhibition assay

The expression vectors (12 μg of full-length spike protein and 6 μg of pMX-GFP) were cotransfected into 293T cells on 100 mm dish using 43.2 μg of Polyethylenimine Max (Polyscience, PA, USA). After two days, cells were washed with PBS supplemented with 0.5% BSA and 2 mM EDTA (staining buffer), incubated with diluted serum samples for 20 min at 4 °C, washed again, and incubated with premixed ACE2-SBP and APC-conjugated streptavidin (0.6 μg/ml and 0.4 μg/ml, respectively) for 20 min at 4 °C. After the final wash, the cells were analyzed by a FACS Verse. The MFI among GFP⁺ cells was calculated using FlowJo (BD) and used as an indicator of Spike-ACE2 binding. Cells without serum and without ACE2-SBP were used as negative and positive controls, respectively. The representative plots are shown in Supplementary Fig. 1D. The measurement procedures are illustrated in Fig. 1C.