Anti cd66b pacific blue

Antibody-dependent neutrophil phagocytosis (ADNP) was determined using an adaptation of a flow cytometry-based phagocytic assay described previously^{41 (link),42 (link)}. Briefly, fluorescent, streptavidin-conjugated microspheres were coated with chemically biotinylated SIV_mac239 gp120 (Immune Technology) for 2 hrs at 37°C and washed with 0.1% BSA in PBS. The antigen-coupled beads were incubated with a 1:100 dilution of plasma for 2 hrs at 37°C and washed with PBS to remove unbound antibodies. WBCs were isolated from healthy donor blood anti-coagulated with acid citrate dextrose by ACK lysis of red blood cells. 5×10⁴ WBCs were added to the opsonized beads and incubated for 1 hr at 37°C and then stained with anti-CD66b-Pacific Blue (Biolegend). Cells were then fixed with 4% paraformaldehyde solution and analyzed on a flow cytometer. A phagocytic score was derived as an integrated MFI by multiplication of the fraction of neutrophils that phagocytosed one or more opsonized beads by the MFI of this population.