Gut Acidification Monitoring Assay: A Technique to Study the Acidification of the Intestinal Lumen in Drosophila using Bromophenol Blue Dye
Transcrição
The insect midgut contains a copper cell region – CCR – which is responsible for gut acidification. This region contains specialized secretory cells – copper cells – that produce acid, which passes through the narrow channel formed by the neighboring interstitial cells. The acid releases into the intestinal lumen to aid digestion.
To monitor gut acidification in Drosophila, take adult flies starved for an adequate duration. Place the flies in a Petri plate containing a single drop of fly food supplemented with bromophenol blue, or BPB – a pH-sensing indicator dye. Allow the starved flies to forage for a fixed period.
After food ingestion, acid produced by the copper cells reaches the intestinal lumen and mixes with the food containing BPB. At an acidic pH, the BPB molecules get protonated, which changes their color from blue to yellow – indicating acidification.
On completion of the experimental duration, place the Petri plate on ice. A low temperature ceases neuromuscular activity in the flies and anesthetizes them. Place an individual fly under a stereomicroscope and surgically isolate the intact digestive tract.
Examine the CCR color to determine the status of gut acidification. A yellow coloration indicates acidification, while a blue coloration indicates the absence of acidification.
Inspect the digestive tract of all the flies, and calculate the percentage of individuals displaying gut acidification.
