An In Vivo Assay to Quantify Bacterial Phagocytosis in Adult Drosophila Flies

Take anesthetized adult Drosophila flies with their ventral side up.

Load a glass microneedle with heat-killed bacteria, surface-labeled with a fluorophore, suspended in colored liquid to aid visibility during the injection.

Inject the suspension into the upper corner of the abdomen, releasing bacteria into the hemolymph — the circulatory fluid in the body cavity.

Hemolymph contains immune cells called hemocytes, a subset of which cluster around the abdominal dorsal vessel — a segment of the circulatory system.

Pattern recognition receptors on hemocytes bind to pathogen-associated molecular patterns in bacteria, causing bacterial phagocytosis.

Rest the flies after the first injection. Then, anesthetize the flies again to inject trypan blue dye into the abdomen.

Under a fluorescence microscope, visualize punctate fluorescence from bacteria phagocytosed by dorsal vessel-associated hemocytes.

Fluorescence emitted by extracellular, non-phagocytosed bacteria is quenched by the injected dye, differentiating them from phagocytosed bacteria.

Obtain the fluorescence intensity ratio of the dorsal vessel to the background, quantifying bacterial phagocytosis.