Measuring Phagocytosis

In mammals, the process of phagocytosis is essential for a variety of biological events, including tissue remodeling and the continuous clearance of dying cells. Phagocytes (for example macrophages, dendritic cells, and neutrophils) engulf and eliminate foreign invaders such as bacterial or viral pathogens. In this article we briefly discuss phagocytosis and its role in the innate immune system response, and we outline the basic principle behind a common assay designed for the detection of phagocytosis.

Phagocytosis represents an early and crucial event in triggering host defenses against invading pathogens. Phagocytosis comprises a series of events, starting with the binding and recognition of particles by cell surface receptors, followed by the formation of actin-rich membrane extensions around the particle. Fusion of the membrane extensions results in phagosome formation, which precedes phagosome maturation into a phagolysosome. Pathogens inside the phagolysosome are destroyed by lowered pH, hydrolysis, and radical attack. These early events that are mediated by the innate immune system are critical for host survival. As a result of this process, pathogen-derived molecules can be presented at the cell surface (antigen presentation), allowing the induction of acquired immunity.

Phagocytosis has been traditionally assayed by measuring the engulfment of a cell "substrate". The most common substrates used in phagocytosis assays are erythrocytes (red blood cells) and zymosan (yeast) particles. When using red blood cells (RBCs) in the assay, the RBCs are first opsonized with serum or IgG; then they are incubated with phagocytes. The RBCs that are not engulfed by the phagocytes are removed, and the phagocytes are then lysed to release the engulfed RBCs. The detection of RBCs can then be quantified using a standard microplate reader. The same assay principle is followed when using the Zymosan substrate; however, since Zymosan is prepared from yeast cell walls consisting of protein carbohydrate complexes, the opsonization step is not needed.