Phagocytosis Assay with Biotinylated Protein

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).