Interaction of non‐adherent suspended neutrophils to complement opsonized pathogens: a new assay

Phagocytosis of opsonized pathogens by circulating neutrophils is essential to host defense, which when overwhelmed contributes to sepsis. To investigate ligation of complement receptors CR3 and CR4 in non‐adherent neutrophils, we designed a novel dual optical trap assay, one holding a suspended unactivated cell and one presenting a specific ligand‐coated bead to the cell surface. Anti‐CD18 coated beads (which mimic the bacterial opsonizing complement fragment iCB3) elicited both pseudopodial protrusion and subsequent phagocytosis. This is in sharp contrast to previously reported responses of adherent neutrophils, which phagocytize opsonized particles without pseudopod formation. We probed actomyosin pathways in the cell□fs pseudopodial and phagocytic response through actin disruption or myosin light‐chain kinase inhibition, which dose‐dependently reduced pseudopod formation and phagocytosis rates. In summary, the new assay can be used to study the response of suspended neutrophils to a variety of ligands, and in a first application, we found that local ligation of CR3/4 in suspended unactivated neutrophils induces local pseudopod formation and phagocytosis via an actomyosin dependent pathway.