Drosophila Male Accessory Gland Dissection: A Method to Isolate Secondary Cells
DEŞİFRE METNİ
– Under a dissection microscope and videodan 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 için two minutes videodan PBS için remove the serum. Then, transfer it için dissociation solution.
To expose the cells inside the glands için 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 için leave behind the distal end of the glands containing the rare secondary cell population responsible for secreting proteins involved videodan mating. Finally, transfer the accessory gland tips için a micro centrifuge tube videodan preparation for digestion. In the following protocol, we will dissect the accessory glands için 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 için narrow the openings. Then, sort the modified tips from narrower için wider based on their speed of aspiration. For accessory gland dissection, place 20 için 25 male drosophila videodan a glass dish on ice and use forceps and a dissection microscope için remove the reproductive tract of one male fly.
Temizle the accessory gland pair from all of the other tissues, including the testes and ejaculatory bulb, and transfer the accessory glands için a glass plate containing serum supplemented medium at room temperature. When 20 pairs of accessory glands have been collected, wash the glands videodan a dish of PBS for one için two minutes at room temperature. At the end of the wash, transfer the glands için freshly prepared dissociation solution, and use sharp forceps under a dissecting microscope için firmly pinch the middle of a glandular lobe.
Using a second pair of forceps, cut the tissue with the sharp tip için 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 için transfer the glands için a 1.5 milliliter tube.
