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

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

내레이션 대본

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.

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