Two methods were used to measure the food intake of Drosophila. One is the excreta quantification (EX-Q) method developed by our lab. Flies were aged on standard/DHM medium until the 10 days, then transferred to the food intake assay medium (Erioglaucine, Sigma 861146, for dye food), and kept on the food intake vials for 24h. Then measured the food intake of flies and the data analysis as the description [51 (link)]. The other one is the “one hour feeding assay”. The control and DHM-treated flies in 10 days were used to measure the food intake as the description [52 (link)].
Erioglaucine
Manufactured by Merck Group
Sourced in United States
Erioglaucine is a laboratory dye used as a colorant in various analytical and experimental applications. It is a water-soluble, blue-green powder that can be used to stain or label samples for visualization and identification purposes. The core function of Erioglaucine is to provide a distinctive coloring agent for various scientific and research procedures.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Tnf α, by Sino Biological (1 mentions)
Antihuman hrp, by Sino Biological (1 mentions)
Streptavidine hrp, by Merck Group (1 mentions)
Allura red, by Merck Group (1 mentions)
Tartrazine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab6994, by Abcam (1 mentions)
Human plasma fibronectin, by Merck Group (1 mentions)
Ab2213, by Abcam (1 mentions)
Ab150080, by Abcam (1 mentions)
Albumin from bovine serum bsa, by Merck Group (1 mentions)
Goat anti mouse igg h l alexa fluor 488, by Abcam (1 mentions)
Primary huvec, by Lonza (1 mentions)
Ab150113, by Abcam (1 mentions)
Ab133567, by Abcam (1 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (1 mentions)
Thp 1 cells, by Korean Cell Line Bank (1 mentions)
Goat anti rabbit igg h l alexa fluor 594, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
10 protocols using erioglaucine
1
Drosophila Food Intake Measurement
2
Nematode Reproduction Assay on Tomato
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Meloidogyne incognita was maintained on tomato cv. UC82B. Nematode eggs were extracted from infected roots in 0.5% NaOCl and eggs were hatched as described in Martinez de Illarduya et al. (2001) [62] (link). Three weeks after agroinfiltration, tomato roots were infected with freshly hatched 1000 infective-stage juveniles and maintained at 24°C. Six weeks later, roots were washed from soil particles, weighed and stained in 0.001% erioglaucine (Sigma). Individual roots were chopped into small pieces, mixed and egg masses were counted in two 10 g subsamples and the average calculated.
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Meloidogyne incognita was maintained on tomato cv. UC82B. Nematode eggs were extracted from infected roots in 0.5% NaOCl and eggs were hatched as described in Martinez de Illarduya et al. (2001) [62] (link). Three weeks after agroinfiltration, tomato roots were infected with freshly hatched 1000 infective-stage juveniles and maintained at 24°C. Six weeks later, roots were washed from soil particles, weighed and stained in 0.001% erioglaucine (Sigma). Individual roots were chopped into small pieces, mixed and egg masses were counted in two 10 g subsamples and the average calculated.
3
Fabrication of Microcrystalline Wax Immunoassay
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Microcrystalline wax (Ibercer 2616, melting point of 81 °C) was kindly provided by Iberceras Specialities S.L.U. (Madrid, Spain). We purchased 100 µm-thick polyester transparency A4 sheets from APLI S.L. (Alaquàs, Spain). Pressure-sensitive-adhesive films (PSA) (ARcare® 8939, 120 µm total thickness) were obtained from Adhesives Research Inc. (Glen Rock, PA, USA). Poly(methyl methacrylate) (PMMA) was acquired from Servicio Estación (Barcelona, Spain). Unbacked 120 µm-thick nitrocellulose was obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Polydimethylsiloxane (PDMS Sylgard 184 kit, which includes the Sylgard 184 silicone elastomer base and the Sylgard 184 silicone elastomer-curing agent) was purchased from Sigma Aldrich (St. Louis, MO, USA). For the immunoassay, the capture (anti-human) and detector antibodies (anti-human-HRP), along with the analyte (TNFα), were obtained from Sinobiological Inc. (Beijing, China). The buffer reagents (Phosphate Buffered Saline (PBS), Tween 20, Bovine Serum Albumin (BSA)), the dyes (Tartrazine, Erioglaucine and Allura red), the streptavidine-HRP, as well as the substrate (3,3′,5,5′-Tetramethylbenzidine (TMB)) were obtained from Sigma Aldrich (USA). Sodium Hydrogen Carbonate (NaHCO3) was purchased from Panreac Química (Barcelona, Spain).
4
Fly Proboscis Extension Assay
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Two-day old flies raised on standard media were transferred to vials containing 0.7% agar after 24 h of fasting. A single fly was transferred by use of an oral aspirator and trapped in a 200 μl pipette tip with the head exposed. 1 μl of sucrose solution (150 mM) mixed with 3% erioglaucine (Sigma Aldrich, St. Louis, MO; 861146) was placed on the edge of the pipette tip within range of the proboscis. Proboscis extension and duration of feeding were observed for approximately 5 min.
5
Vascular Cell Coculture Protocol
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Primary HASMCs, primary HUVECs, SMC growth medium-2 (SmGM-2), and endothelial cell growth medium-2 (EGM-2) were supplied from Lonza (Basel, Switzerland). THP-1 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). RPMI 1640 and Dulbecco’s modified Eagle’s medium (DMEM) were ordered from Corning (New York, NY, USA), and human plasma fibronectin was purchased from Millipore (Burlington, MA, USA). Sixteen percentage formaldehyde and Hoechst 33342 were obtained from Thermo Fisher Scientific. Trichloro(1H,1H,2H,2H-perfluorooctyl)silane, erioglaucine, Triton X-100, and albumin from bovine serum (BSA) were bought from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-smooth muscle myosin heavy chain 11 antibody (ab133567, 1:25), mouse anti-CD31 antibody conjugated with Alexa Fluor 488 (ab215911, 1:100), mouse anti-intercellular adhesion molecule 1 (ICAM1) antibody (ab2213, 1:50), rabbit anti-von Willebrand factor antibody (ab6994, 1:100), goat anti-mouse IgG H&L Alexa Fluor 488 (ab150113, 1:200), and goat anti-rabbit IgG H&L Alexa Fluor 594 (ab150080, 1:200) were ordered from Abcam (Cambridge, UK). Recombinant human TNF-α was supplied from PeproTech (Rocky Hill, NJ, USA). A PDMS polymeric base and a curing agent were purchased from Dow Chemical Company (Midland, MI, USA).
6
Quantifying Dye Uptake in Fly Guts
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Blue dye (Erioglaucine, 861146, Sigma-Aldrich) was mixed with the diet to a concentration of 0.16%. Flies were allowed to feed on the diet with blue dye for 24 h. Then, they were homogenized in 200 µl PBS, and the number of adults used for homogenization was noted. The homogenate was centrifuged at 17,800 g for 10 min and 90 µl of the supernatant was dispensed into each well of a 96-well plate. The absorbance at 625 nm was measured and the obtained value was normalized according to the number of guts used.
7
Smurf Assay for Gut Barrier Integrity
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The integrity of the gut barrier was tested by placing flies on blue dyed lifespan analysis food prepared with 2.5% (w/v) Erioglaucine (Sigma-Aldrich), also known as FD&C blue no 1. Female flies were set up as described in the lifespan analysis section. Flies were kept on blue food for 24 h and the Smurf phenotype was scored for another 24 h. Two independent experiments were carried out. The Smurf assay has been described previously in recent publications [25 ,47 (link)]. Smurf data were analyzed with Fisher´s exact test for significance using MS Excel and XLSTAT. All values are given in S4 Table .
8
PDMS-based Fluorescence and Colorimetric Quantification
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The quantification of fluorescence and colorimetric assays was performed
with the double-layer PDMS. Solutions of erioglaucine and fluorescein
isothiocyanate (FITC) were obtained from Sigma-Aldrich and MK Science
(Seoul, Korea), respectively, and used to characterize the colorimetric
or fluorescence intensity in a microchannel. Each solution was diluted
with distilled water for the preparation of 2×, 3×, and
4× diluted solutions. For the fluorescence reading, the image
of the FITC-filled microchannel was taken by a charge-coupled device
(CCD) camera (DP72; Olympus, Tokyo, Japan) attached to a fluorescence
microscope (LX72; Olympus). For the colorimetric reading, the image
of an erioglaucine-filled microchannel was taken by a stereomicroscope
(SZX16; Olympus) equipped with the CCD camera. The images were further
analyzed by ImageJ software. For colorimetric analysis, the percentage
of blue pixels out of overall red, green, and blue (RGB) pixels was
calculated according to the following equation:
with the double-layer PDMS. Solutions of erioglaucine and fluorescein
isothiocyanate (FITC) were obtained from Sigma-Aldrich and MK Science
(Seoul, Korea), respectively, and used to characterize the colorimetric
or fluorescence intensity in a microchannel. Each solution was diluted
with distilled water for the preparation of 2×, 3×, and
4× diluted solutions. For the fluorescence reading, the image
of the FITC-filled microchannel was taken by a charge-coupled device
(CCD) camera (DP72; Olympus, Tokyo, Japan) attached to a fluorescence
microscope (LX72; Olympus). For the colorimetric reading, the image
of an erioglaucine-filled microchannel was taken by a stereomicroscope
(SZX16; Olympus) equipped with the CCD camera. The images were further
analyzed by ImageJ software. For colorimetric analysis, the percentage
of blue pixels out of overall red, green, and blue (RGB) pixels was
calculated according to the following equation:
9
Nanoparticle Synthesis and Historical Pens
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Silver nitrate, trisodium citrate, ascorbic acid and the reference dyes Eosin Y, erioglaucine, and crystal violet were purchased from Sigma-Aldrich and used without further purification. All glassware was cleaned with aqua regia and deionized water prior to nanoparticle synthesis. Historical felt-tip pens belonging to the film director Federico Fellini were made available by the studio Fellini in Cinecitta' (Italy). Contemporary felt-tip pens were purchased from local stores.
10
Larval Dye Uptake Assay
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3rd instar larvae (10 total) were collected and placed on cotton wool soaked with solution of 4.5% dissolved yeast granules and 0.5% Erioglaucine (Sigma, 861146). Controls contained no dye. Feeding was allowed for 2 hours at 25°C. 5 larvae per tube were crushed in 100μL of double distilled water. Solution was spun at 14000 rpm for 15 minutes and 50μL was withdrawn for absorbance measurement at 625nm in a 96-well plate. 5μL was used to measure protein content using the Pierce BCA Protein Assay kit (#23227).
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