Our protocol provides an effective and repeatable approach to evaluate and quantify phagocytic events in hemocytes. The main advantage of this technique is that we can quantify phagocytic events within individual hemocytes which allows us to detect variations in phagocytic processes among genotypes in individual flies. Performing dissections can be challenging, so when performing this technique for the first time, practice dissections as much as possible before carrying out the full procedure.
Begin by preparing glass needles for injection. Fill a one millimeter syringe with mineral oil, and attach the 30 gauge hypodermic needle provided with the nano-injector. Insert the 30 gauge needle into the blunt end of a pulled capillary needle and fill it with mineral oil.
Slowly remove the 30 gauge needle making sure that no air bubbles are formed. Remove the collet and place the sealed O-ring with the indentation facing up. Place the larger O-ring onto the metal plunger, then reattach the collet without tightening.
Insert the metal plunger into the blunt end of the oil-filled glass needle and gently push it down inserting it into the larger O-ring. Then tighten the collet until secure. After filling the needle with fluoro or pH-sensitive particles, inject the flies in the sternopleural plate of the thorax.
The injection is successful if the green dye is going into the fly. Place the injected flies into a new food vial noting the time that the first and last fly were injected. Lay the vial on its side until all flies have recovered to prevent them from getting stuck in the food.
Orient the fly ventral side up under a dissecting stereomicroscope. Then insert an insect pin into the thorax and another through the most posterior end of the abdomen near the genitalia. Once all flies have been pinned, use a transfer pipet to add enough dissection media to cover the flies.
Remove the head with cuticle scissors, and make a horizontal incision directly above the posterior pin in the abdomen, then another at the most anterior end of the abdomen. Make a vertical incision connecting the two horizontal incisions which will open up the abdominal cavity. Use forceps to remove the internal organs and tissue, making sure to avoid the dorsal vessel.
If necessary, use additional pins to secure the cut ends of the cuticle. Then remove the thorax with the cuticle scissors. Discard the dissection media and replace it with one milliliter of 4%PFA, protecting the dissections from light as much as possible.
Fix the dissections for 15 minutes at room temperature while rocking at 20 rpm. Then remove the fixative and add one milliliter of 1X PBST. If desired, stain the dissections with antibodies.
After mounting the cuticles on a microscope slide, use forceps to orient them ventral side up, and make sure that the dorsal vessel is visible. Young and aged flies were assessed for age-specific ability to carry out phagocytosis by fluorescent imaging of hemocytes along the dorsal vessel. An antibody against pericardin was used to ensure that only cells along the dorsal vessel were counted.
Since fluorescently-labeled E.coli particles are one micrometer in length and hemocytes are 10 micrometers in diameter, only fluorescent events within a 10 micrometer diameter centered on a DAPI-positive nucleus were counted. ImageJ software was then used to quantify the events. When attempting this procedure, it's important to visualize that the fly was actually injected and to remember that timing is critical.
Completing injections and dissections in a timely manner will help minimize experimental error and possible variations in phagocytic rate. This technique will allow researchers to explore a variety of research topics including questions regarding cellular immunity, different populations of blood cells or distinguishing phagocytosis from AMP production during a successful immune response.
