A cell-free scintillation proximity assay for studies on lysosome-to-phagosome targeting

Abstract

Phagocytes, such as macrophages, neutrophils, and dendritic cells, play important roles in the innate immune system through their ability to engulf, kill, and digest invading microbes. In cooperation with the humoral adaptive immune system, coating of substrates with immunoglobulin G (IgG) antibodies enhances several aspects of phagocytosis, including the recognition of substrates by cell surface IgG (Fcgamma) receptors, particle internalization, generation of microbicidal oxygen species, and targeting of lysosomes to phagosomes. We describe a cell-free scintillation proximity assay developed to study the mechanisms of lysosome targeting to phagosomes and the regulation of this process by IgG. The approach involves the use of isolated phagosomes containing scintillant latex beads and lysosomes labeled with a tritiated marker. Scintillation results only when lysosomes and phagosomes come into immediate contact and requires supplementation of reactions with adenosine triphosphate and cytosol; addition of cytosol from IgG-conditioned cells enhances this signal. The method is useful for investigating the biochemistry and regulation of the early tethering and docking steps of lysosome and phagosome interactions.