Phagocytosis assay kit

In 24-well plate, 400 µl of u-THP-1 (5 × 10⁵ cells/ml) cells in the presence of PMA were seeded in each well. Phagocytosis assay kit (Cell Biolabs Inc., San Diego, CA) was used according to the manufacturer’s protocol. Briefly, IgG₁ (Cat#:sc-52003) or SLAMF7 agonist antibody (10 µg/ml) (Cat#:NBP2-45868) was added to the cell cultures and incubated for 15 min followed by addition of 40 µL of an E. coli suspension from the kit. Negative control (without E. coli) was also included. Plate was incubated for another 6 h. After incubation, cells in the wells were washed with cold serum-free RPMI medium, followed by addition of a fixation solution and a blocking and permeabilization solution with washes between each step and addition of substrate followed by a stop solution. Absorbance (405 nm) was measured at five different time points (0, 5, 10, 15 and 30 min) using SpectraMax M5 microplate reader (Molecular Devices, San Jose, CA).

RAW 264.7 cells were adjusted to 5 × 10⁴/ml, cultured for 24 h and starved for 12 h. The cells were then pre‐treated with HBP vacuoles in various concentrations of (0.001, 0.005 and 0.01) μg/ml for 6 h. Cells were then treated with Zymosan suspension provided by Phagocytosis assay kit (Cell biolabs) for 2 h, and the experimental procedure was carried out according to the product manual. pVMA11 vacuoles and phagocytosis inhibitor (Cytochalasin D) were used as controls.