Alexa fluor 647 conjugated donkey anti human igg fc f ab 2 fragment

Expi293F cells (ThermoFisher Scientific) were grown in Expi293 expression medium (ThermoFisher Scientific). Cell surface display DNA constructs for the SARS-CoV-2 spike variants together with a plasmid expressing blue fluorescent protein (BFP) were transiently transfected into Expi293F cells using ExpiFectamine 293 reagent (ThermoFisher Scientific) per manufacturer’s instruction. Two days after transfection, the cells were stained with primary antibodies at 10 μg/mL concentration. For antibody staining, an Alexa Fluor 647 conjugated donkey anti-human IgG Fc F(ab’)2 fragment (Jackson ImmunoResearch, West Grove, PA) was used as secondary antibody at 5 μg/mL concentration. Cells were run through an Intellicyt iQue Screener Plus flow cytometer. Cells gated for positive BFP expression were analyzed for antibody and ACE2₆₁₅-foldon T27W binding. The flow cytometry assays were repeated three times with essentially identical results.