100ul of 2% Bromophenol blue sodium (pH indicator, Sigma), or 5% sucrose ± 80 mM paraquat (methyl viologen, Sigma) was added to a food vial and pipet tip was used to poke 4–6 holes in the food to allow full absorption. Flies were fed for overnight. For Lactobacillus feeding, flies were fed 400 ul of concentrated bacteria (OD100, in 5% sucrose) mixed with either 100ul 2% pH indicator or 2.5% blue dye (FD&C Blue 1, SENSIENT) in filter paper for overnight. During dissection, the fly head and posterior cuticle was left intact to prevent dye leakage. Images were taken immediately after each gut was dissected. Long-term exposure of flies with pH indicator on CO2 pad will disturb normal pH patterns. Edu incorporation The Click-iT EdU Imaging Kit from Invitrogen was applied. Briefly, 100ul EdU solution (100uM, diluted with water from 10mM stock) was added to a food vial (pipet tip was used to poke 4–6 holes in the food to allow full absorption of the solution). Flies were fed EdU food at 29°C for 4 days.
Bromophenol blue sodium
Manufactured by Merck Group
Bromophenol blue sodium is a laboratory dye commonly used as a pH indicator. It exhibits a blue color at pH values above 4.6 and a yellow color at pH values below 3.0. This dye is often used in various analytical and biochemical applications to monitor pH changes or to track the progress of electrophoresis and chromatography experiments.
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5 protocols using bromophenol blue sodium
1
Dye-Based Gut pH and Cell Proliferation
2
Midgut pH Change Detection
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The Bromophenol blue assay was used to detect the pH change in the midgut as previously described (Li et al., 2016 ). Briefly, 100 µl of 2% Bromophenol blue sodium (Sigma, B5525) was added to the food surface and several holes were poked to allow the Bromophenol blue solution completely absorbed by the food. Images were taken immediately after 12 h.
3
Feeding Drosophila with pH Indicator
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Putting 150ul of 2% Bromophenol blue sodium (pH indicator, Sigma) was added to a food vial, and stir the blue fuel with a sterile rod. Flies were fed for 10 h. For E. faecium feeding, flies were fed 200 ul of 1 OD bacteria (in 5% sucrose) mixed with 100ul 2% pH indicator in filter paper for 24 h.
4
Feeding Drosophila with pH Indicator
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Putting 150ul of 2% Bromophenol blue sodium (pH indicator, Sigma) was added to a food vial, and stir the blue fuel with a sterile rod. Flies were fed for 10 h. For E. faecium feeding, flies were fed 200 ul of 1 OD bacteria (in 5% sucrose) mixed with 100ul 2% pH indicator in filter paper for 24 h.
5
Bromophenol Blue Midgut pH Assay
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The bromophenol blue assay was performed as previously described to reveal the pH change in the midguts (Li et al., 2016 (link)). One hundred microliters of 2% Bromophenol blue sodium (Sigma, B5525) were added to the food surface. Then, several holes were poked to allow the Bromophenol blue solution full absorption. Twelve hours later, images were captured after dissection.
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