Drosophila Male Accessory Gland Dissection: A Method to Isolate Secondary Cells

– Under a dissection microscope and in 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 세스 two minutes in PBS 세스 remove the serum. Then, transfer it 세스 dissociation solution.

To expose the cells inside the glands 세스 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 세스 leave behind the distal end of the glands containing the rare secondary cell population responsible for secreting proteins involved in mating. Finally, transfer the accessory gland tips 세스 a micro centrifuge tube in preparation for digestion. In the following protocol, we will dissect the accessory glands 세스 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 세스 narrow the openings. Then, sort the modified tips from narrower 세스 wider based on their speed of aspiration. For accessory gland dissection, place 20 세스 25 male drosophila in a glass dish on ice and use forceps and a dissection microscope 세스 remove the reproductive tract of one male fly.

Clear the accessory gland pair from all of the other tissues, including the testes and ejaculatory bulb, and transfer the accessory glands 세스 a glass plate containing serum supplemented medium at room temperature. When 20 pairs of accessory glands have been collected, wash the glands in a dish of PBS for one 세스 two minutes at room temperature. At the end of the wash, transfer the glands 세스 freshly prepared dissociation solution, and use sharp forceps under a dissecting microscope 세스 firmly pinch the middle of a glandular lobe.

Using a second pair of forceps, cut the tissue with the sharp tip 세스 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 세스 transfer the glands 세스 a 1.5 milliliter tube.