Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

The overall goal of the following experiment is to isolate a cell population that is enriched for intestinal stem cells from the mid guts of young and old drosophila using fluorescence activated cell sourcing. This is achieved by first dissecting the mid guts of flies in which the cells of interest express GFP In the second step. The mid guts are dissociated into single cell suspensions and then sorted by facts based on differences in their GFP intensity.

Ultimately, this gating strategy allows the enrichment for and isolation of intestinal stem cell populations from young and old drosophila. Mid guts. Being able to isolate intestinal stem cells from young and from old mid guts of adult drosophila will facilitate the identification of novel molecules in these cells that function during aging.

The implications of this technique ultimately extend towards the development of molecular therapies aiming to improve regeneration and organ maintenance during aging. The dissection and tissue dissociation procedure will be demonstrated by Helen Talc, a graduate student from my laboratory, and the fact sorting will be demonstrated by GaN a graduate student from the Institute of Immunology Before beginning the dissection. Anesthetize one vial of 40 flies with carbon dioxide on a standard fly bed.

Then use a razor blade to decapitate all of the flies and transfer them to a dissection dish coated with agarro gel. Pour enough cold PBS plus BSA into the dish to cover the agros. The flies will float then for each insect.

In turn, grab a fly abdomen with one pair of forceps and the thorax with another. Separate the thorax from the abdomen. The gut will be visible and the crop will most likely still be connected to the thorax.

Grab the gut and pull it outta the thorax. Then grab the crop and pull the gut slightly anteriorly away from the abdomen to unfold it. Next, use one of the forceps to grab the posterior end of the abdomen and the other to grasp the edge of the anteriorly open cuticle.

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. Remove the four gut malian tubes, hind gut, and ovaries leaving the bare midgut as the mid guts are isolated. Use a glass pasta pipette to periodically transfer several of the intestinal tissues at a time to a 1.5 milliliter micro centri tube containing cold PBS plus BSA on ice within two hours of the dissection.

Remove the solution from the samples and add 500 microliters of 0.5%tripsin EDTA solution to the mid guts. Vortex each sample for 20 seconds, then incubate the mid guts with gentle rocking at 20 RPM at room temperature. After 25 to 30 minutes, vortex the samples again and then let the intact midgut tissue sink to the bottom of the tube.

Once the tissue has settled, use a flamed glass pasta pipette to carefully remove the cells in suspension and filter them through a 35 micrometer nylon mesh into a fresh 1.5 milliliter micro centrifuge tube. After straining centrifuge, the cells note that at the beginning of the digest, a cell palette may not be visible, but will become visible as tissue digestion progresses. Carefully transfer the tripsin supinate back to the original sample tube containing the remaining intact midgut tissue and gently resuspend the pellet in 400 microliters of cold PBS plus BSA place the micro centrifuge tube containing the dissociated cells on ice protected from light.

Then vortex the tripsin solution containing the remaining intact midgut tissue and place the samples back onto the rocker for another 30 minutes. At room temperature, hold the dissociated cells periodically until all of the midgut tissue has been digested. Then spin down the pooled cells and carefully remove or but the last 50 to 100 microliters of the supernatant.

Gently resuspend the pellet in 800 microliters of PBS and BSA plus ox, and then filter the cells through a 35 micrometer nylon mesh cell strain a snap cap into a five milliliter round bottom Falcon tube on ice, protected from light at least one hour. Before sorting switch on the fax machine. Choose the 70 micrometer nozzle size to inject the cells into the sheath fluid stream and make sure that the fluidic system is free of air bubbles.

Adjust the amplitude of the core fluid stream so that the gap value corresponds to the reference value and let the core fluid stream stabilize. Then set the parameters for the cell sorting. Load the dissociated cells obtained from wild type guts and adjust the forward and side scatter voltages To plot the cells in the center of the scatter plot.

Adjust the fite voltage so that all the cells are plotted below 10 to the two and the logarithmic x axis to set the fit E parameter limit for the autofluorescence. Then load the dissociated cells obtained from the wild type guts with cyt added. Adjust the Pacific blue voltage value in the forward scatter a plot, and then gate the living cells from the dead.

Cytx positive cells. Load the dissociated cells obtained from the escargo GAL four U-A-S-G-F-P fly line without cyt and adjust the fitzy voltage to ensure that all of the GFP positive cells are plotted within the scatter plot. Then load the dissociated cells obtained from the Escargo GAL four U-A-S-G-F-P fly line with cyt added and adjust the Pacific blue voltage value.

Use these parameters to gate to the living cytx negative cells. To identify the GFP positive cells. Set the size scatter A and forward scatter a gait according to the size and granularity of the GFP positive cells.

Then set the forward scatter h and w gait to exclude the GFP positive cell doublets based on their size and the side scatter h and w gait to exclude the doublets based on their granularity. Now, use the side scatter h and w gait to depict the GFP positive cells in a histogram plot that shows the number of cells versus the GFP intensity. Two distinct peaks of GFP positive cells will be visible.

Create a gate for each of the peaks. The P five gate will contain the cell population that is enriched for the intestinal stem cells. The P six gate will contain the cell population that is enriched for the eblasts.

The cells contained in each gate can be sorted separately. Then back gate in a contour plot to verify that the two distinct GFP positive cell populations contain living cells of the correct size. Finally, use the two-way purity sort mode to sort the cells into 1.5 milliliter micro centrifuge tubes containing 600 microliters of RNA later solution at a low flow rate.

Immediately after the sort is finished, briefly vsx the sorted cells and to keep the micro centrifuge tubes on ice until proceeding with the RNA isolation or other downstream applications. To validate the purity of the isolated intestinal stem cells and enter oblasts, a posts sort analysis can be performed. For example, in this experiment, the posts sorted intestinal stem cell and enter eblast Populations exhibited purities between 95%and 98%To verify that the GFP low cell population is enriched in intestinal stem cells, a reverse transcriptase PCR for the notch ligand delta can be performed as shown here.

Delta expression is stronger in the cells present in the GFP low population than in the GFP high cells with tubulin serving as a loading control. The same gating strategy can be used when sourcing intestinal stem cells derived from old mid guts. Note that in old mid guts, the two peaks containing the GFP low and the GFP high cells remain distinctly separated during aging.

It can be concluded from these findings that sourcing for GFP positive cells by facts allows the enrichment of intestinal stem cells and enter oblasts from young and old mid guts. While attempting this procedure, it's important to execute the steps in a timely manner. Taking care to sort the cells directly after the tissue dissociation procedure and to set the facts parameters correctly, using the appropriate controls For further validation of intestinal stem cell identity.

An additional reporter construct can be tested that should only be active in the progenitor cells, but not in the intestinal stem cells.