FACS Analysis With the HEK293T Cells

Two million HEK293T cells were seeded in a 100 mm cell culture petri-dish the day before transfection. Twelve ug of the plasmid (pCMV14-3X-Flag-SARS-CoV-2 S) was transfected with a 1:1 ratio of lipofectamine 2000 (Thermo Fisher scientific). After 48 h, the cells were gently washed with 1xdPBS and were harvested in FACS buffer (1xPBS + 10%FBS +1mM EDTA). Cells were washed in FACS buffer two to three times with gentle centrifugation (250 g) for 5 min at RT. Cells were resuspended in 3 ml of FACS buffer and the 0.1-0.2x10⁶ cells were aliquoted in a 1.5 ml microcentrifuge as per the requirement. Each tube was incubated with 20 ug/ml of primary antibody (pooled mouse serum IgG from anti-RBD, anti-RBD+AddaVax, anti-RBD+imject, and control group was used as primary antibody) for 1 h at RT followed by washing with FACS buffer three times; here untransfected cells were also used as the untransfected mock control. Further cells were incubated with florescence dye conjugated secondary antibody (Alexa Fluor 488) at 1:400 dilution, followed by washing with FACS buffer and fixing with 4% PFA for 10 min at RT. Samples were acquired in BD FACSCanto II and raw data were analyzed using FlowJo software.