Quantification of RBD Binding to Cells

The binding of recombinant RBD (Acrobiosystems Cat#: SPD-C82E9) to THLE-2 and Vero E6 cells, as well as to primary hepatocytes, was measured as described in ref. ^{27 (link)}. Briefly, cells were harvested from culture plates using cell dissociation buffer (Thermo Fisher Cat#: 13151-014), counted and 100,000 cells were distributed in 96-well polystyrene conical bottom plates (Thermo Fisher Cat#: 249570). After washing cells with blocking buffer (PBS containing 0.5% BSA; Sigma Aldrich Cat#: A9647), they were incubated with biotinylated RBD (20 μg/mL) for 40 min on ice. After incubation, cells were washed again with blocking buffer and incubated for an additional 15 min with streptavidin-PE (1:200, Thermo Fisher Cat#: 12-4317-87) on ice in a total volume of 100 μL of blocking buffer. Finally, cells were washed twice and resuspended in a blocking buffer containing DAPI (Invitrogen Cat#: D1306) to discriminate alive cells. All centrifugation steps were performed at 300×g for 5 min at 4 °C. Cells were acquired on a FACSymphony cytometer (BD Biosciences) and results were analyzed using FlowJo version 10 (BD Biosciences).