Mouse anti flag m2 fitc

Human embryonic kidney HEK293T cells stably expressing the SARS-CoV-2 spike glycoprotein S1 (generated by transfection with plasmid DNA encoding for S1 protein and subsequent clonal selection) were seeded in 96-well microtiter plates (5 × 10⁵ cells/well) pretreated with polyethyleneimine (Sigma-Aldrich). After 24 h, cells were washed with PBS, fixed with 4% formaldehyde, permeabilized with PBS/Tween (PBS, 0.2% Tween 20), and blocked with 2% BSA in PBS/Tween. Cells were then incubated with 50 μL/well scFv at concentration of 10 μg/mL, for 1 h at 4°C. After three washings with PBS/Tween, plates were treated with 10 μg/mL of a mouse anti-FLAG M2-FITC (Sigma-Aldrich). Cells were ultimately counterstained with Draq5 (Axxora) and analyzed for fluorescence using the high-content screening system Operetta (PerkinElmer).

Human embryonic kidney HEK293T cells stably expressing the SARS-CoV-2 spike glycoprotein S1 (generated by transfection with plasmid DNA encoding for S1 protein and subsequent clonal selection) were washed with PBS and incubated with 360 nM scFv for 1 h at 4°C. After three washings with PBS, cells were treated with 10 μg/mL of a mouse anti-FLAG M2-FITC (Sigma-Aldrich), washed again, and analyzed for fluorescence using an ATTUNE NxT acoustic focusing cytometer (Invitrogen/Thermo Fisher Scientific). Dead cells were ruled out from the analysis using propidium iodide staining (P4864; Sigma Aldrich).