This is the fastest and right protocol to monitor the Drosophila gut acidification with robust and authentic results. This method will help to determine the role of gut acidification in multiple organisms. And this phenomenon is The simplicity of this technique is the main advantage.
As such, it can be utilized for the non-model organisms. This method can monitor the acid release for many systems including the cell lines and 3D on live cultures. This technique is simple with minimal effort.
Food containing Bromophenol blue should be prepared fresh. In addition, while dissection gut should not be stretched. Start arranging for the gut acidification monitoring assay with the preparation of the fly food with pH sensing Bromophenol blue or BPB dye.
To do so, melt the fly food in a microwave and let it cool until lukewarm. Add one milliliter of 4%BPB to one milliliter of the lukewarm food with a mixing well. Using a pipette, add the fly food containing BPB into a single dot of approximately 200 microliters in the center of a petri dish.
Next, collect zero to two-days-old non-virgin female Drosophila melanogaster flies, and allow the flies to recover on standard corn meal food. Before the experiment, starve the flies for 24 hours at room temperature in a vial containing a laboratory wipe tissue soaked with approximately two milliliters of deionized water. Transfer starved flies into a petri dish containing single dots of the freshly prepared fly food, supplemented with 2%BPB, to allow the flies to forage for four hours at room temperature while exposed to light.
After four hours collect the flies, then proceed to surgically isolate the guts of the anesthetized flies in 1X PBS with forceps under a stereo microscope. Count the number of the flies that show robust BPB standing in the guts, and calculate the percentage using the equation. Following dissection, mount the samples in PBS onto a glass slide and place the prepared slide under the microscope.
Then adjust the focus using the IPS. Once done, shut off the IPS to open the shutter for the camera. Following gut dissection, some Drosophila melanogaster female flies were found to produce acid as indicated by the yellow color in the CCR of the gut.
Whereas the intestines of some flies were blue suggesting a failure of the flies to acidify their guts. Within 30 minutes, approximately 20%of flies had acidified the gut. After an hour, 40%of the gut guts showed acidification, while after two to three hours of feeding, the percentage of acidified guts increased to 60 to 70%Almost 90 to 95%of the guts were acidified when flies were fed for four hours.
No difference in gut acidification was observed for two different temperature conditions. The efficiency of the gut acidification protocol was assessed in various Drosophila species. All the tested species showed robust gut acidification, suggesting the protocol can be applied to other organisms.
The most important thing to remember, is to place the star flies into the petri plate with yeast and Bromophenol blue for four hours. This technique will help to understand the basic biology and cellular pathway involved in the gut acidification. It should also help to identify the drug target to create the diseases related to the gut acidification.
