Trogocytic Assay for SARS-CoV-2 Spike Antibodies

HEK-293T cells (Invitrogen) were transfected using SARS-CoV-2 S expression plasmid expression vector and lipofectamine (thermofisher) in OptiMEM as previously described.⁸⁶ (link) SARS-CoV-2 S expression HEK293T cells were stained with PKH26 dye according to the manufactures protocol (Sigma-Aldrich). Next, the stained HEK293T cells were incubated for 30 min at 37°C with serial antibodies dilutions. 2G12-IgG1, specific for HIV-1 gp120, was used as a negative control. After incubation, the cells were washed and THP-1 cells (ATCC), stained with carboxyfluorescein succinimidyl ester according to manufactures protocol (Thermofisher), were added to the HEK293T cells at a 2:1 effector:target cell ratio. The plates were spun down for 30 sec to promote cell to cell contact before incubation for 1 hours at 37°C. After incubation, the plates were washed twice, resuspended in PBS 2% fetal calf serum (FCS) and analyzed using flow cytometry. Trogocytic activity was calculated by the mean fluorescence intensity (MFI) of the double positive PKH26+ CFSE+, THP-1 cells and depicted as the area under the curve.