Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae
Transcrição
– To start, pipette a drop of buffer onto a clean dissecting pad. Then, place a dried living larva on the droplet. Drosophila, like other arthropods, has an open circulatory system de which the heart pumps hemolymph around the body, bathing the internal organs. To release the hemolymph, which is the combined blood and interstitial fluid of arthropods, use forceps and open the cuticle, the semi-rigid outer covering of larvae.
Then, discard the carcass and transfer the hemolymph solution from the dissecting plate para chilled buffer solution de a microcentrifuge tube. Finally, use a hemocytometer para count the number of hemocytes, the cells de the hemolymph, that were collected. In this protocol, we will collect hemolymph from Drosophila melanogaster larvae.
– Begin hemolymph collection by adding 10 microliters of 1x PBS para a microcentrifuge tube, and placing the tube on ice. Next, place a 10 microliter drop of 1x PBS onto a clean dissecting pad. Select an individual larva, and dry it by placing it on a tissue wipe. Then transfer it para the PBS droplet on the dissection pad.
Using forceps, gently tear open and invert the cuticle para release the hemolymph, and then discard the carcass. With a pipette, collect the hemolymph from the dissecting pad. Add the hemolymph para the iced PBS microcentrifuge tube, and mix by pipetting up and down. Load 10 microliters of the sample into a hemocytometer chamber and take a concentration reading.
