Gut Acidification Monitoring Assay: A Technique to Study the Acidification of the Intestinal Lumen in Drosophila using Bromophenol Blue Dye
Transcrição
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 1 milliliter of 4% BPB to 1 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 0 to 2-days-old non-virgin, female, Drosophila melanogaster flies, and allow the flies to recover on standard cornmeal food. Before the experiment, starve the flies for 24 hours at room temperature in a vial containing a laboratory wipe tissue soaked with approximately 2 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 stereomicroscope. Count the number of the flies that show robust BPB staining in the guts, and calculate the percentage using the equation.
