Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

This protocol is an excerpt from Reimels and Pfleger, Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae, J. Vis. Exp. (2016).

1. Circulating Hemocyte Concentration

1. To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 – 6 hr.
2. Collect larvae in dissecting dish wells filled with 1x phosphate buffered saline (PBS).
3. For each larva, place 10 µl 1x PBS in a microcentrifuge tube on ice and 10 µl 1x PBS on a clean dissecting pad. Place the dissecting pad on an illuminated stereomicroscope base.
4. Dry an individual larva by placing it on a tissue wipe before transferring it to a PBS drop on the dissection pad.
5. Using a pair of forceps, gently tear open and carefully invert the cuticle to release the hemolymph.
   NOTE: Take care not to scrape or jab the cuticle as this could potentially release sessile hemocytes.
6. Using a pipet, collect the hemolymph from the dissecting pad. Avoid collecting larval debris such as the fat body.
7. Add the hemolymph to a microcentrifuge tube and mix by pipetting up and down.
   NOTE: Several samples can be collected at once and kept on ice for up to an hour.
8. Optional: Mix Trypan blue with the hemolymph sample in equal parts to dye and exclude dead cells. Count cells within 3 min of adding Trypan blue because it is toxic to cells.
9. Mix the sample by pipetting up and down before loading 10 µl into a hemocytometer chamber.
10. If using an automated cell counter, set appropriate parameters for cell size and circularity. For example, set a minimum cell size of 2 µm, a maximum cell size of 22 µm, and a circularity of 75 – 80% roundness to detect normal, round hemocytes. Experiment with different parameters to detect larger and spindle-shaped hemocytes, such as lamellocytes. Alternatively, use a hemocytometer to manually count hemocytes.