Ligand-induced clustering of SIRPα on phagocytes. Freshly isolated human neutrophils and monocytes as well as undifferentiated THP-1 cells and PMA-differentiated THP-1 cells were labeled (at RT, without fixation) with antibody P3C4 against human SIRPα (A-D). Bound antibody was detected with R-phycoerythrin-tagged secondary antibody. Inset images magnify the membrane association. (E-F) Neutrophils and monocytes were also stained with biotinylated Ig-CD4718 and fluorescent extravidin. Ligand-induced SIRPα clusters are clearly visible for both ligands and for all phagocytes. Average pixel intensity in unclustered regions was calculated to determine the fraction of cell area with at least 2- or 5-fold the mean unclustered pixel intensity. Antibody-mediated preclustering of SIRPα with P3C4 enhances Ig-CD47 binding (G).
