A fluorescence technique to distinguish attached from ingested erythrocytes and zymosan particles in phagocytosing macrophages.

While investigating the role of [Ca2+]i in Fc receptor-mediated phagocytosis, we found that clamping the [Ca2+]i to low levels by a variety of methods often led to increased plasma membrane permeability, rendering the hypotonic lysis method for calculating the phagocytosis index unreliable. To overcome this difficulty we developed a method for calculating the phagocytosis index of IgG-coated RBC that does not depend on hypotonic lysis. It depends on the ability of ingested RBC to create dark-appearing phagocytic vacuoles when the cytoplasm of murine macrophages is stained with acridine orange. Uningested or partially ingested RBC do not change the appearance of the otherwise uniformly stained cytoplasm of the macrophages. Since this method does not rely on hypotonic lysis to distinguish attached from ingested RBC, it can be used in situations when macrophages are unusually sensitive to hypotonic treatment (e.g. after clamping the macrophage [Ca2+]i to low levels). It can also be applied to the study of phagocytosis of particles that are not susceptible to hypotonic lysis (e.g., zymosan).