Dissection of the Midgut from an Adult Fly: A Method for Isolating the Digestive Tract

Overview

In this video, we highlight a dissection of the adult Drosophila intestine and collection of midguts suitable for single-cell dissociation and FACS in order to isolate intestinal stem cells.

Protocol

This protocol is an excerpt from Tauc et al., Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging, J. Vis. Exp. (2014).

NOTE: If this protocol is used for the first time to isolate ISCs by FAC sorting, the following controls are mandatory in order to initially set the FACS parameters properly: Dissociated cells from wild type (e.g. w¹¹¹⁸) Drosophila midguts without Sytox. Dissociated cells from wild type (e.g. w¹¹¹⁸) Drosophila midguts with Sytox (see Step 3.6). Dissociated cells from midguts of the esg-Gal4, UAS-GFP fly line without Sytox.

1. Preparation of Solutions and Dishes for Gut Dissection

1. Prepare 4-6 vials each containing 40 female flies from the esg-Gal4, UAS-GFP fly line.
2. From a 10x PBS stock solution prepare 500 ml 1x PBS (1.8 mM NaH₂PO₄·H₂O, 8.4 mM Na₂HPO₄·2H₂O, 175 mM NaCl, adjust pH to 7.4).
3. Prepare a 3.5% agarose solution in 1x PBS (3.5 g electrophoresis grade agarose in 100 ml 1x PBS). Prepare dissection plates by pouring this solution into petri dishes (diameter 8.5 cm) to cover the bottom. The agarose layer prevents damaging the tips of the forceps during the dissection procedure. After the gel has solidified store the dissection plates at 4 °C.
4. Prepare 300 ml of 1x PBS + 1% BSA fresh before dissecting and place the bottle on ice or at 4 °C.
5. Turn on centrifuge and let it cool to 4 °C.
6. Flame the tips of 2 glass Pasteur pipettes to smoothen the edges.
7. Clean two pairs of forceps and one razor blade with 70% ethanol.
8. Place four to six 1.5 ml microcentrifuge tubes for collecting dissected midguts on ice.

2. Preparation of the Gastrointestinal Tract and Dissection of the Midgut

1. Anesthetize flies from one vial (40 flies) with CO₂ on a standard fly bed, decapitate all flies using a razor blade and transfer them to the dissection dish. Pour cold 1x PBS/1% BSA solution into the dissection dish to cover the agarose gel. The flies will float.
2. Grab the fly abdomen with one pair of forceps, while holding the thorax with the other pair of forceps. Separate the thorax from the abdomen. The gut will be visible and the foregut/crop will most likely still be connected to the thorax.
3. Grab the gut and pull it out of the thorax. To unfold the gut, grab the crop and pull the gut slightly anteriorly away from the abdomen.
4. Grab the posterior end of the abdomen with one pair of forceps and the edge of the anteriorly open cuticle with the other pair of forceps. Be careful not to destroy the protruding gut tissue. Pull the posterior end away to break the cuticle and continue pulling posteriorly very gently until the entire gut has been pulled out of the abdominal cavity. The crop may have to be removed beforehand if it is too large to fit through the body cavity.
5. Remove the foregut, Malpighian tubules, hindgut and ovaries leaving the bare midgut.
6. Using a glass Pasteur pipette, transfer the batch of dissected midguts to a 1.5 ml microcentrifuge tube containing cold 1x PBS/1% BSA solution and keep the tube on ice. The samples must be processed within 2 hr.
7. After the dissection of an entire batch is complete, rinse the dissection dish with double distilled water to wash off left over debris. Start dissecting the next batch of midguts (repeat Steps 2.1–2.7). Dissect as many batches as possible within 2 hr, then proceed to Step 3.1.

3. Digestion of the Gut Tissue to Harvest Cells for FAC Sorting

1. Remove the 1x PBS/1% BSA solution from the midguts and add 500 µl of 0.5% Trypsin-EDTA solution to each sample.
2. Vortex well for 20 sec and incubate the samples by gentle rocking/rotating at 20 rpm at room temperature for 25-30 min.
3. Vortex again after about 30 min and let the intact midgut tissue sink to the bottom of the tube. Carefully remove the cells that are in suspension with a flamed glass Pasteur pipette and filter them through a 35 µm nylon mesh into a fresh 1.5 ml microcentrifuge tube.
4. Spin the cells down at 100 x g for 5 min at 4 °C. Of note, when starting the digest, a cell pellet might not be visible at first but will become visible as the tissue digest progresses.
   1. Carefully transfer the Trypsin solution back to the original sample tube containing the remaining intact midgut tissue. Avoid transferring too many cells from the pellet.
   2. Gently re-suspend the cell pellet in 400 µl of cold 1x PBS/1% BSA. Keep the microcentrifuge tubes containing the dissociated cells on ice and cover the tubes with aluminum foil to protect the cells from light.
   3. Vortex the Trypsin solution containing the remaining intact midgut tissue again and place the samples onto the rocker for another 30 min at room temperature.
5. Repeat steps 3.3 to 3.4.3 until all of the midgut tissue has been digested. Combine the dissociated cells from all microcentrifuge tubes into one microcentrifuge tube. This may require a centrifugation step as in 3.4, since the volume of all cell suspensions combined may exceed 1.5 ml. Keep the cell suspension on ice and protect the cells from light.
6. Spin the dissociated cells down at 100 x g for 5 min at 4 °C. Carefully remove as much of the supernatant as possible leaving approximately 50-100 µl of the 1x PBS/1% BSA solution in the microcentrifuge tube. Gently resuspend the dissociated cells in 800 µl 1x PBS/1% BSA containing Sytox (1:20,000).
7. Transfer the cell suspension to a 5 ml round bottom Falcon tube by filtering the cells through a cell strainer snap cap (35 µm nylon mesh). Always keep cell samples on ice and protected from light. The cells are now ready to be sorted.
8. Pipette 600 µl of RNAlater solution into each microcentrifuge tube into which the cells will be sorted for subsequent RNA isolation. For other downstream applications the cells can be collected in a different solution, e.g. in sterile PBS.

Materials

Name	Company	Catalog Number	Comments
Forceps: Dumont, Inox Biologie #5	Fine Science Tools	11252-20
SefarNitex 03-150um/38 (35 µm nylon mesh)	Sefar	3A03-0150-102-00
Falcon 5 ml Round Bottom Polystyrene Test Tube with Cell Strainer Snap Cap	Corning	352235
Polymax 1040	Heidolph	543-42210-00
Albumin from bovine serum (BSA)	Sigma	A4503-50G
0.5% Trypsin-EDTA	Invitrogen	15400-054	Trypsin obtained from a different company most likely has a different activity and the duration of the trypsin digest has to be adjusted accordingly.
SYTOX Blue Dead Cell Stain for flow cytometry	Life Technologies	S34857
RNAlater Stabilization Solution	Life Technologies	AM7023	Other solutions, e.g., Trizol can be used for subsequent RNA isolation
FACSAria II cell sorter	Becton Dickinson		Turn on one hour prior to sorting