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

This protocol is an excerpt from Immarigeon et al., A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands, J. Vis. Exp. (2019).

1. Solutions and Material Preparation

1. Prepare the following solutions.
   1. Prepare Serum Supplemented Medium (SSM): Schneider's Drosophila medium complemented with 10% heat inactivated fetal bovine serum and 1% Penicillin-Streptomycin.
   2. Prepare aliquots of 1x trypsin enzyme (e.g., TrypLE Express Enzyme) and store at room temperature.
   3. Prepare aliquots of papain [50 U/mL] (stored at -20 °C and thawed only once).
   4. Prepare 1x phosphate buffer saline (PBS, stored at room temperature).
2. Prepare multiple flame-rounded pipet tips for the physical dissociation of accessory glands.
   NOTE: Use low retention tips for handling accessory glands as they tend to adhere to untreated plastic. The process of flame-rounding reduces the tip opening and smoothens the edges of the tip. This ensures an efficient dissociation after peptidase digestion without shearing the cell membranes.
   1. Cut one 200 µL tip with a sharp blade and gently pass it over a flame to round out its tip so that the opening is wider but smooth for handling during step 2.7.
   2. Narrow the opening of multiple 1,000 µL tips by passing the tip opening near a flame for less than one second. Rotate the tip slightly to avoid over-melting or clogging. Sort the tips made from narrower to wider. This can be done by timing the speed of aspiration. Try to dissociate a small scale sample of AGs to test the efficiency of the narrowest tips.
      NOTE: These tips are critical for mechanical agitation. Quality flame-rounded pipet tips allow for complete dissociation while preserving cell viability. As such, these tips are washed thoroughly with water at the end of the procedure for reuse. This allows for day to day reproducible dissociation.

2. Dissection of Accessory Glands

1. Put 20 to 25 male Drosophila in a glass dish on ice.
2. Dissect 1 male in SSM. Take off its reproductive tract and clear the accessory gland pair from all other tissues apart from the ejaculatory duct.
   NOTE: Remove testes because the released sperm can create clumps and disturb the dissociation process. Remove the ejaculatory bulb, which makes handling difficult when it floats.
3. With forceps, transfer accessory glands to a glass plate filled with SSM at room temperature. Repeat steps 2.2-2.3 20 times to obtain a batch of 20 pairs of glands in SSM.
   NOTE: These steps will be achieved in 15 to 20 min. AGs should look healthy, and the peristaltic movements of the muscle layer around glands should be visible. GFP can be monitored using a fluorescent microscope.
4. Transfer accessory glands to 1x PBS for a 1-2 min wash at room temperature.
   NOTE: For better dissociation, it is imperative to rinse the glands with PBS. The duration of this step, however, should be limited as the cells show signs of stress in PBS; dissociated secondary cells in PBS rapidly swell and die.
5. Dilute 20 µL papain [50 U/mL] into 180 µL of 1x trypsin enzyme to obtain the dissociation solution (to scale up or down keep 9 µL of trypsin enzyme and 1 µL of papain for each male). With forceps, transfer accessory glands to this solution.
6. Isolate the tip of accessory glands (containing secondary cells) from the proximal part. Use fine forceps to pinch firmly the middle of a glandular lobe and cut with the sharp tip of a second forceps. Remove the proximal part of accessory glands and the ejaculatory duct to improve dissociation and reduce cell sorting time.
   NOTE: Dissecting the distal part from 20 pairs of glands will take 15 to 20 min and digestion by peptidases will thus start at room temperature.
7. After all gland tips have been dissected, transfer them into a 1.5 mL tube using a special 200 µL pipet tip prepared at Step 1.2.1. It should be wide, rounded and wet prior to handling glands.
   NOTE: To pipet accessory gland tissue, tips should always be pre-wet with appropriate solution (trypsin enzyme or SSM, keep one tube of each for this purpose). Carefully rinse tips between samples to avoid contamination.