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