This in vivo phagocytosis assay allows researchers to carry out genetic screens and genome-wide association studies to identify novel genes that regulate phagocytosis in adult blood cells. This experiment is quantitative, easy to perform, and can be applied to the screening of live animals for host factors that influence pathogen recognition, uptake, and clearance. After pulling thin-wall glass capillaries with a needle puller, use a micrometer to hold the needle under a microscope, and use number five, 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 1.6 milligrams per milliliter 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 in 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 flies 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. After being mounted ventral side down on a piece of electrical tape, the first two segments of the abdomen, where the dorsal vessel is located, are clearly visible. Key sources of experimental error arise at the injection and imaging steps of the procedure.
Using the same needle to inject multiple flies may cause it to become clogged with fly tissue or particles. Flies that do not receive enough Trypan Blue fluorescence brightly throughout their entire abdomen, which can reduce the ratio of the dorsal vessel to the background fluorescence, thus decreasing the true fluorescence intensity ratio of the animal. Flies should be photographed while immobilized by carbon dioxide, as actively moving flies produce blurry images that cannot be quantified.
A mutant line from the Zuker collection of ethyl methanesulfonate-treated flies are unable to phagocytose gram-negative and gram-positive bacteria and displays almost no dorsal vessel fluorescence in the in vivo phagocytosis assay, paired to the isogenic background Zuker strain and another common laboratory control strain. Keep track of the time and the order in which flies are injected to ensure that flies are at comparable stages of pathogen recognition and uptake when imaged. The molecular mechanisms underlying phagocytic defects can be determined by studying single hemocytes with confocal microscopy or after fluorescence-activated cell sorting or by magnetic bead isolation of adult hemocytes.
