Flow cytometry–based antibody binding assay

Antibody binding to full-length SARS2-S epitope mutants on the cell surface was measured by flow cytometry. HEK-293T cells were seeded at a density of 2.5 × 10⁵ cells/ml in a T25 flask. After reaching 80% confluency, cells were transfected with an expression plasmid encoding full-length SARS2-S mutants with a C-terminal Flag tag, using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were dissociated by cell dissociation solution (Sigma-Aldrich, Merck KGaA; catalog no. C5914). To detect total spike expression, cells were permeabilized by 0.2% saponin and subjected to anti-Flag tag antibody staining. For cell surface antibody binding measurement, intact (nonpermeabilized) cells were incubated with 20 μg/ml of 47D11, ACE2-Fc, CR3022 (target SARS2 RBD core), 49F1 (target SARS2-S1 outside RBD), and anti-Flag (Sigma-Aldrich, F1804) for 1 hour on ice, followed by incubation with 1:200 diluted Alexa Fluor 488–conjugated goat anti-human IgG antibodies (Invitrogen, Thermo Fisher Scientific, #A-11013) or goat anti-mouse IgG antibodies (Invitrogen, Thermo Fisher Scientific, #A28175) for 45 min at RT. Cells were subjected to flow cytometric analysis with a CytoFLEX flow cytometer (Beckman Coulter). The results were analyzed with FlowJo (version 10). FSC/SSC gates were used to select mononuclear cells. Control antibody staining was used to define positive/negative cell populations.