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.

Use a micrometer to hold the needle under a microscope, and use number 5 fine-point stainless steel tweezers to break the tip to a 100-micrometer tip diameter. To measure the volume of liquid that will be injected into each fly, load a capillary needle with sterile 5% food coloring in PBS, and expel the liquid onto a drop of mineral oil on a 0.01-millimeter stage micrometer.

Dispense 10 microliters of 1.6mg/ml particles onto a small square of parafilm, and pull the liquid into the needle. Mount the needle into the injector nozzle, and line the anesthetized flies along their designated area on the fly pad, ventral side up, with the heads oriented toward the front of the pad.

Place the vials and corresponding areas on the bench, and inject the flies at the upper corner of the abdomen with five 100-millisecond pumps of liquid to deliver about 10 nanoliters of particles total. Transfer each fly into the appropriate vial as it is injected, noting the time on the vial.

Next, load a new needle with 0.4% trypan blue solution, and set the pneumatic injector to "Gated", to allow a constant flow of air to push the liquid out of the needle. 30 minutes after the initial injection, inject each fly abdomen with trypan blue until the abdomens are full and distended. Mount the flys on microscope slides with electrical tape, ventral side down, pushing the wings to the side of the fly to secure them to the tape. Then, gently push the head into the tape to ensure that the fly will not move.

Immediately after all of the flies have been secured, image the insects, one at a time, at a 25 or 32 times magnification on an inverted fluorescence microscope attached to a digital camera and computer, focusing on the dorsal vessel of each fly using the computer software for the digital camera. Then, record the exposure time and magnification between experiments.

To quantify the fluorescence, open an appropriate imaging analysis program, and open one image. To measure the fluorescence intensity of the dorsal vessel, draw a polygon around the dorsal vessel, and select "Measure" to record the fluorescence intensity inside the polygon.

To determine the background fluorescence intensity, copy the first polygon, and move it to an area adjacent to the dorsal vessel of each fly. Then, select "Measure", and record the fluorescence intensity of the background area.

To normalize the dorsal vessel fluorescence by the background fluorescence, after measuring the fluorescence intensities of the rest of the flies, divide the dorsal vessel fluorescence by the background fluorescence, and calculate the average normalized dorsal vessel fluorescence intensity of all the flies in one strain.