Drosophila Male Accessory Gland Dissection: A Method to Isolate Secondary Cells
Transcrição
– Under a dissection microscope and de serum supplemented media, remove the reproductive tract from each fly with forceps. First, remove parts of the digestive tract that may have accompanied the reproductive tract. To isolate the accessory glands and ejaculaory duct, remove the testes, the ejaculatory bulb, and the terminalia. Wash the tissue for one para two minutes de PBS para remove the serum. Then, transfer it para dissociation solution.
To expose the cells inside the glands para the dissociation solution, hold the center of each gland with forceps and slice through the gland with a pair of sharp forceps. Remove the proximal portion of the gland and the ejaculatory duct para leave behind the distal end of the glands containing the rare secondary cell population responsible for secreting proteins involved de mating. Finally, transfer the accessory gland tips para a micro centrifuge tube de preparation for digestion. In the following protocol, we will dissect the accessory glands para isolate secondary cells for transcriptome analysis.
– Before beginning the procedure, cut and flame round, wide 200 microliter tips for handling the glands, and pass the tip opening of multiple 1000 microliter tips near a flame for less than one second para narrow the openings. Then, sort the modified tips from narrower para wider based on their speed of aspiration. For accessory gland dissection, place 20 para 25 male drosophila de a glass dish on ice and use forceps and a dissection microscope para remove the reproductive tract of one male fly.
Limpar the accessory gland pair from all of the other tissues, including the testes and ejaculatory bulb, and transfer the accessory glands para a glass plate containing serum supplemented medium at room temperature. When 20 pairs of accessory glands have been collected, wash the glands de a dish of PBS for one para two minutes at room temperature. At the end of the wash, transfer the glands para freshly prepared dissociation solution, and use sharp forceps under a dissecting microscope para firmly pinch the middle of a glandular lobe.
Using a second pair of forceps, cut the tissue with the sharp tip para separate the proximal region of the accessory gland and the ejaculatory duct from the portion of the gland containing secondary cells. Then, use a pre-wet flame rounded wide 200 microliter pipette tip para transfer the glands para a 1.5 milliliter tube.
