Quantifying Phagocytic Bead Internalization

To ensure that phagocytosis measured by flow cytometry resulted in actual bead uptake, and not bead attachment alone, we performed imaging flow cytometry to assess the amount of bead attachment and internalization. Immune complexes were formed with HIVIG, IVIG and no antibody control as described above. WBCs were incubated with immune complexes at 4 °C and 37 °C for 1 h. Cells from 12 wells were then pooled, washed and stained with CD66b-AF647 (Biolegend) and DAPI (Invitrogen). Bead attachment and internalization by CD66b⁺ cells were visualized using an ImageStreamX MkII (EMD Millipore) across 500,000 independent neutrophil events (60× objective, 405, 488 and 642 nm lasers, with extended depth of focus) and quantified using IDEAS 6 software utilizing the internalization module. An internalization feature was employed describing the ratio of fluorescence intensity of the beads inside the cell against whole cell intensity, with positive scores representing greater internalization.