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