Phagocytosis assay

Before the day of assay:

1. Prepare Drosophila eggs in advance to have early 3rd instar larvae on the day of assay. Ideally, have them in food plates, where larvae can be easily removed.

On the day of assay:

1. Prepare needles for injection by pulling borosilicate glass 3.5” capillaries (e.g. Drummond Scientific Co. 3-000-203-G/X) in a needle puller (e.g. Narishige PC-10).
2. Cut the needle tip with a razor to make an opening.
   Tip: Do not make a blunt cut, try to have an opening with a 45° angle. This will make it easier for the needle to go trough the larval cuticle. At this point the needle opening can be small, as long as the oil and injection solutions can go trough it easily.
3. Backfill needle with mineral oil (e.g. Sigma-Aldrich #M5904) with a syringe and attach it to a nano-injector (e.g. Drummond Scientific Co. Nonoject II), according to manufacture specifications.
4. Eject the excess of mineral oil.
5. Fill the needle with pHrodo Red E.coli Bioparticles (1mg/ml; Molecular Probes).
   Tip: If large aggregates are formed in the pHrodo solution, use a sonicator to break them apart.
6. Carefully remove larvae with forceps from cornmeal food and place them aligned in a filter paper, in groups of 10.
   Tip: Before starting with injections, try to pierce trough the cuticle of a larva with the needle. If the needle bends too much, cut it a little bit more. Repeat the process until the needle does not bend but it is still thin enough to not create a big wound.

7. Under a dissecting microscope, use a forcep to hold the larvae in place with one hand and hold the nano-injector with the other hand. Inject larvae with 69nl of pHrodo. Try to inject the larvae in the posterior 1/3 of body length, from the side.

8. Move the filter paper to inject the next larvae until all 10 are injected.
9. After injection, add ddH20 with a brush next to the larvae, remove them carefully with forceps and transfer them into a drop of yeast or cornmeal food.
10. Incubate larvae for 1h hour.
11. Clean larvae thoroughly with Ringer’s solution before transferring them into 20μl Ringer’s solution on a dissection well plate in groups of 10 larvae.
12. Bleed larvae by tearing the cuticle from the ventral side with forceps and transfer the hemocyte solution into a slide.
13. Allow hemocytes to settle for 20min at room temperature in a humid chamber.
14. Mount a cover slide and take pictures immediately after incubation period.

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