Thp 1 effector cells

HEK293F cells (Invitrogen) at a density of 1 × 10⁶cells/mL were transfected using SARS-CoV-2 S plasmid and PEImax (1 µg/µl) in a 3:1 ratio in OptiMEM. HEK293F cells were harvested 72 h after transfection and their plasma membrane was stained with 10 µM PKH26 (Sigma-Aldrich) dye in PBS, for 20 min (RT) with periodic mixing. Cells were washed twice with PBS and taken up in culture medium. THP-1 effector cells (ATCC) were stained intracellularly with 0.05 µM carboxyfluorescein succinimidyl ester (CFSE, ThermoFisher) in PBS and incubate 20 min (RT) with periodic mixing. Cells were washed twice with PBS and taken up in culture medium.). PKH26 stained HEK293F cells were opsonized for 30 min at 37 °C, with serial MAb dilutions. 2G12-IgG1, specific for HIV-1 gp120, was used as a negative control. After incubation, cells were washed and THP-1 cells were added to the HEK293F cells at a 2:1 effector:target ratio. Plates were centrifuged shortly to promote cell to cell contact and incubated 1 h at 37 °C. Afterwards, cells were washed and resuspended in PBS/2% FCS. Flow cytometry was used to measure the double positive, PKH26⁺CFSE⁺THP-1 cells. ADCT was calculated by the fraction of THP-1 cells that received membrane fragments from the HEK293F cells. To exclude measurement of antibody-independent trogocytosis, cells were gated on stained HEK293F and THP-1 cells in the absence of antibodies.

This assay was performed as described previously.⁶³ (link) In short, Fluorescent NeutrAvidin beads (Thermo Scientific) were incubated overnight at 4°C with biotinylated GPC-I53-50A protein (10μg/5μL beads suspension). After incubation, the beads were washed twice using PBS 2% bovine serum albumin (BSA). 50 μL of the bead suspension were placed in a V-bottom 96-well plate and incubated with 10-fold serial dilutions of guinea pig serum at a start dilution of 1:1000 in PBS 2%BSA. After 2 h at 37°C, the plates were washed and 5×10⁴ THP-1 effector cells (monocytes; ATCC) were added to each well. To promote beads to cell contact, plates were quickly spun down before incubation for 5 h at 37°C. After incubation, the cells were washed and resuspended in PBS 2% FCS. Cells were analyzed by flow cytometry and the phagocytic activity was determined by the area under curve of the MFI (beads positive cells x mean MFI FITC).