Phagocytosis Assay Using Biotinylated SE36

SE36 was biotinylated by ECL Protein Biotinylation Module (GE healthcare) and then the biotinylated SE36 was coated on 1 μm yellow-green fluorescent (505/515 nm) neutravidin beads (Thermo Fisher Scientific). IgG was purified using the Melon Gel IgG Purification kit (Thermo Fisher Scientific) and re-suspended in PBS. The VTN-depleted sera were prepared by anti-VTN IgG-immobilized column using AminoLink Plus Immobilization Kit according to the manufacturer's instructions (Thermo Fisher Scientific). Phagocytosis assays were performed as previously described in ref.^{54 (link)}, with minor adjustments. In brief, 5 × 10⁴ THP-1 cells were added to each well in a final volume of 100 μL, and the plate was incubated overnight under standard tissue culture conditions (37 °C, 5% CO₂). After incubation, 5 × 10⁵ beads were placed in each well of flat-bottom 96-well plates and incubated for 6 h. Cells were washed in ice-cold PBS by centrifugation at 500 g for 5 min at 4 °C. In each experiment, at least 10,000 events were recorded using FACSCalibur flow cytometer (FCM) and analysed using CellQuest Pro Version 5.2.1 software (BD Biosciences, Franklin, NJ, USA). The phagocytosis index (PI) (%) was calculated from the ratio of median fluorescent intensity (MFI) of THP-1 cells that engulfed tested beads/MFI of THP-1 cells that engulfed SE36-beads × 100.