A Hemocyte Disturbance Assay to Assess Hemocyte Re-Adhesion to Hematopoietic Pockets

Begin with a tube containing glass beads and second instar Drosophila larvae expressing fluorescently-labeled hemocytes — insect's blood cells comprising circulating and resident hemocytes.

The resident hemocytes adhere to hematopoietic pockets — a niche for blood cell generation.

Mechanically, disturb the larvae to release resident hemocytes from the hematopoietic pockets into the circulatory system.

During recovery, the receptors of resident hemocytes interact with the hematopoietic pocket and re-adhere.

Post-recovery, place one larva into a specialized glass slide well and create incisions on the ventral side at the anterior and posterior ends, releasing circulating hemocytes.

Wash to remove remaining circulatory hemocytes; transfer the same larva into a new well, and scrape to dislodge the resident hemocytes.

Under a fluorescence microscope, enumerate the released circulating and resident hemocytes.

As recovery time increases post-disturbance, a decrease in circulating hemocytes and an increase in resident hemocytes compared to the start of recovery confirm its re-adherence to hematopoietic pockets.

To mechanically disturb resident hemocytes, select up to four to eight larvae and place them in a 2-milliliter microcentrifuge tube with approximately 0.5 grams of 600-micron glass beads and 0.5 milliliters of water. Vortex the tube by hand at speed 10 for one minute.

Retrieve the larvae from the glass beads by spilling the contents of the microcentrifuge tube into a Petri dish, and picking out the larvae with a paintbrush. For the recovery phase, place larvae in the previously prepared Petri dishes with a thin layer of fly food. Incubate the larvae for 45 minutes to allow the larvae to re-establish their hemocyte pattern. After the recovery period, continue with the bleed-scrape dissections.