Facsymphony cytometer

Cells were thawed in Complete RPMI and stained with fluorochrome-tagged surface antibodies and Ghost-UV450 for 30 min on ice. Using eBioscience™ Transcription Factor Staining Buffer Set, cells were fixed, permeabilized, and stained with intracellular antibodies for 30 min on ice followed by acquisition on the BD FACSymphony cytometer. Gates were set using single-stained and fluorescence minus one (FMO) controls. Data were analyzed using FlowJo10.7.1. Supplementary Table 2a lists cytometry antibodies.

Single-cell suspensions from in vitro cultures or processed animal tissues were stained with a fixable viability dye for 30 min, followed by blocking in 50 µl of 5% FBS in PBS for 20 min at 4°C. Cells were washed and pelleted by centrifugation at 300×g for 10 min at 4°C and stained with 100 µl of antibody cocktail mix at 4°C for 30 min in FACS buffer containing 2% FBS and 20 mM of sodium azide (Sigma-Aldrich) in PBS. The cells were then washed twice in FACS buffer, fixed with 1% paraformaldehyde at pH 7.2–7.4, and resuspended in FACS buffer before being acquired in a BD LSR Fortessa cytometer or BD FACSymphony cytometer. Data were analyzed using FlowJo™ v10.8.1 software. The FACS antibodies and staining reagents used are described in Supplementary Table S1. Appropriate isotype controls and fluorescent minus one (FMO) controls were used to define the expression of certain markers.

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).