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 to two minutes in PBS to remove the serum. Then, transfer it to dissociation solution.

To expose the cells inside the glands to 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 to 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 to a micro centrifuge tube in preparation for digestion. In the following protocol, we will dissect the accessory glands to 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 to narrow the openings. Then, sort the modified tips from narrower to wider based on their speed of aspiration. For accessory gland dissection, place 20 to 25 male drosophila in a glass dish on ice and use forceps and a dissection microscope to 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 to 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 to two minutes at room temperature. At the end of the wash, transfer the glands to freshly prepared dissociation solution, and use sharp forceps under a dissecting microscope to firmly pinch the middle of a glandular lobe.

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