We measured fly TAG and carbohydrates as described previously (Song et al., 2017a (link); Song et al., 2017b (link); Song et al., 2010 (link); Song et al., 2014 (link)). Briefly, 10 flies from each group were homogenized with 1 mL PBS containing 0.2% Triton X-100 using Multi-sample tissuelyser-24 (Shanghai Jingxin Technology) and heated at 70°C for 5 min. The supernatant was collected after centrifugation at 12,000 X g for 10 min at 4°C. 10 μL of supernatant was used for protein quantification using Bradford Reagent (Sigma, B6916-500ML). Whole body trehalose levels were measured from 10 μL of supernatant treated with 0.2 μL trehalase (Megazyme, E-TREH) at 37°C for 30 min using glucose assay reagent (Megazyme, K-GLUC) following the manufacturer’s protocol. We subtracted the amount of free glucose from the measurement and then normalized the subtracted values to protein levels in the supernatant. To measure whole body triglyceride levels, we processed 10 μL of supernatant using a Serum Triglyceride Determination kit (Sigma, TR0100), subtracted the amount of free glycerol in the supernatant from the measurement, and then normalized to protein levels in the supernatant.
E treh
Manufactured by Megazyme
The E-TREH is a laboratory instrument used for the enzymatic determination of trehalose. It provides a precise and reliable method for the quantitative analysis of trehalose in various samples.
Automatically generated - may contain errors
Lab products found in correlation
K gluc, by Megazyme (4 mentions)
Serum triglyceride determination kit, by Merck Group (2 mentions)
Bradford reagent, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Infinity glucose hexokinase reagent, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2 homogenizer, by Qiagen (1 mentions)
Triglycerides reagent, by Thermo Fisher Scientific (1 mentions)
Free glycerol reagent, by Merck Group (1 mentions)
D glucose assay kit, by Megazyme (1 mentions)
Spectramax paradigm plate reader, by Molecular Devices (1 mentions)
5 protocols using e treh
1
Quantification of Fly Metabolites
2
Carbohydrate-rich Diet Protocol
3
Quantifying Metabolite Levels in Drosophila
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Protocols for protein, lipid, and carbohydrate measurements were performed as previously described (Kwon et al., 2015 (link); Ding et al., 2021 (link)). Four female flies were used for each replicate and a minimum of three replicates were measured for each sample group. Flies were homogenized in 200 ul 1X PBS with 0.1% Triton-X and Zirconium 1 mm Oxide Beads (Next Advance Lab Products, ZROB10) using TissueLyser II homogenizer (QIAGEN). Homogenate was incubated at 70°C for 10 minutes and the supernatant was collected after centrifugation at 3,000 g for 5 min. 5 ul of supernatant was applied to Pierce™ BCA Protein Assay Kit (Thermo Scientific, 23227) for detecting protein levels. TAG and free glycerol levels were quantified from 20 ul supernatant using Triglycerides Reagent (Thermo Fisher Scientific™ - TR22421) and Free Glycerol Reagent (Sigma-Aldrich, F6428), respectively. Free glycerol was subtracted from TAG values. Glucose levels were measured from 10 ul supernatant using Infinity Glucose Hexokinase Reagent (Thermo Fisher Scientific™ - TR15421) or D-Glucose assay kit (Megazyme, K-GLUC). Trehalose levels were measured as for glucose but incubated with 0.4 ul trehalase (Megazyme, E-TREH). The amount of glucose was subtracted from trehalose read values. TAG, free glycerol, glucose, and trehalose levels were normalized to corresponding protein levels of each sample.
4
Quantification of Fly Metabolites
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We measured fly TAG and carbohydrates as described previously (Song et al., 2017a (link); Song et al., 2017b (link); Song et al., 2010 (link); Song et al., 2014 (link)). Briefly, 10 flies from each group were homogenized with 1 mL PBS containing 0.2% Triton X-100 using Multi-sample tissuelyser-24 (Shanghai Jingxin Technology) and heated at 70°C for 5 min. The supernatant was collected after centrifugation at 12,000 X g for 10 min at 4°C. 10 μL of supernatant was used for protein quantification using Bradford Reagent (Sigma, B6916-500ML). Whole body trehalose levels were measured from 10 μL of supernatant treated with 0.2 μL trehalase (Megazyme, E-TREH) at 37°C for 30 min using glucose assay reagent (Megazyme, K-GLUC) following the manufacturer’s protocol. We subtracted the amount of free glucose from the measurement and then normalized the subtracted values to protein levels in the supernatant. To measure whole body triglyceride levels, we processed 10 μL of supernatant using a Serum Triglyceride Determination kit (Sigma, TR0100), subtracted the amount of free glycerol in the supernatant from the measurement, and then normalized to protein levels in the supernatant.
5
Quantifying Trehalose Levels in Flies
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Whole-body trehalose levels were measured from five to six groups (each group has four female flies). Fly samples were homogenized with 300 µl TBST buffer (5 mM Tris-HCl [pH 6.6], 137 mM NaCl, 2.7 mM KCl, 0.1% TritonX-100), heated at 75°C for 10 min, and centrifuged at 3000 g for 1 min. Ten µl of supernatant was added to 100 µl glucose assay reagent (Megazyme; K-GLUC) with or without trehalose (1:500; Megazyme; E-TREH) at 37°C for 30 min. The absorbance at 340 nm was measured on a Molecular Devices SpectraMax Paradigm plate reader. The trehalose concentration in the sample was determined by subtracting the glucose concentration from the total sugar concentration.
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