The TAG content quantification was performed using the Triglycerides Reagent (Thermo Scientific, 981786). The assay is based on the enzymatic cleavage of the fatty acid moieties from the glycerol backbone of mono-, di-, and triacylglycerols and the subsequent quantification of the released glycerol. A detailed description of the method can be found at23 (link). Monoacylglycerols are negligible in various insects24 (link) and Drosophila23 (link),25 (link) and only very little diacylglycerol and free glycerol amounts are present, which are negligible in comparison to the triacylglycerol amounts23 (link),25 (link). In this study, we actually also quantified free glycerol levels, however, a fluorometric assay was used given the extremely low levels of free glycerol (see below). In brief, the assay was performed by transferring an aliquot of the supernatant of the above mentioned homogenate (25 µL) to a 96 well plate (Sarstedt, 82.1581) followed by a dilution with 25 µL 0.05% Tween 20 (1:2). Samples and standard were incubated at 37 °C with the Infinity reagent and the absorbance was measured at 510 nm. The total amount of TAG was determined using a glycerol standard (Sigma Aldrich, G7793).
Triglycerides reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Triglycerides Reagent is a laboratory product used for the quantitative determination of triglyceride levels in biological samples. It provides the necessary reagents and materials to measure triglyceride concentrations through a colorimetric or enzymatic assay process.
Automatically generated - may contain errors
Lab products found in correlation
Triglyceride standard, by Horiba (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
G7793, by Merck Group (1 mentions)
Free glycerol reagent, by Merck Group (1 mentions)
Tissuelyser 2 homogenizer, by Qiagen (1 mentions)
Infinity glucose hexokinase reagent, by Thermo Fisher Scientific (1 mentions)
E treh, by Megazyme (1 mentions)
D glucose assay kit, by Megazyme (1 mentions)
Glycerol standard, by Merck Group (1 mentions)
Gum tragacanth, by Merck Group (1 mentions)
Dm2000 upright photomicroscope, by Leica (1 mentions)
Ezrmi 13k, by Merck Group (1 mentions)
Sfa 1, by ZenBio (1 mentions)
Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
13 protocols using triglycerides reagent
1
Quantifying Triglycerides in Insects
2
Serum and Fecal Triglycerides Quantification
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Serum TAG levels were measured using the Triglycerides Reagent (Thermo Scientific, #TR22421) and Triglyceride Standard (Pointe Scientific, #T7531STD). In brief, 2.5 μl of the standard or serum sample and 250 μl of the reagent were added to each microplate well. After incubating at 37 °C for 10 min, absorbance at 500 nm was measured using a Tecan plate reader.
For fecal TAG, feces were collected and homogenized in H2O (1/24, w/v). Chloroform/methanol (2/1, v/v) was then added to extract TAG. 300ul of the bottom chloroform layer containing TAG was transferred to a new tube and dried under nitrogen. The TAG content was measured using Triglycerides Reagent as described above.
For fecal TAG, feces were collected and homogenized in H2O (1/24, w/v). Chloroform/methanol (2/1, v/v) was then added to extract TAG. 300ul of the bottom chloroform layer containing TAG was transferred to a new tube and dried under nitrogen. The TAG content was measured using Triglycerides Reagent as described above.
3
Liver Triglyceride Measurement Protocol
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The total triglycerides in liver were analyzed by Thermo Scientific™ Triglycerides Reagent (TR22421), following the manufacturer protocol. The triglycerides in liver were extracted using the previously described protocol (25 (link)). The triglyceride results were normalized to the mass of the liver tissue initially used for assay, as previously reported (26 (link)).
4
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.
5
Triglyceride Quantification Protocol
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For the determination of the triglyceride levels in the samples, we used the Triglycerides Reagent (Thermo Scientific). We transferred 50 µL of the samples and a serial dilution (1:2 in 0.05% Tween 20 in water) of the glycerol standard (Sigma Aldrich) to a 96-well plate and added 200 µL of the Triglycerides Reagent. The samples and the standard were incubated 45 minutes at 37 °C and the absorbance was read at 510 nm.
6
Cardiac Triglyceride Quantification and Visualization
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Hearts were harvested and flash frozen in an embedding medium containing a 3:1 mixture of Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC) and gum tragacanth (Sigma, St. Louis, MO). 0.3% Oil Red O (BDH, Poole, United Kingdom) staining was performed on 5 μm-thick sections according to standard procedures53 (link). Cardiac sections were visualized with a Leica DM2000 upright photomicroscope. Total triglyceride content was determined biochemically from myocardial tissue collected immediately after euthanasia. Colorimetric quantification of total triglycerides was performed using Triglycerides Reagent—Thermo Fisher Scientific according to the manufacturer’s instructions17 (link),54 (link).
7
Metabolic Markers in Serum and Liver
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Fed serum levels were used to measure insulin (#EZRMI-13K; Millipore), leptin (#90030; Crystal Chem), adiponectin (#KMP0041; Thermo Fisher), and free fatty acids (#sfa-1; ZenBio), and FGF21 (#MF2100; R&D Systems). Insulin levels during glucose tolerance tests were also measured by ELISA (Millipore). Hepatic triglyceride (TG) content was quantified by Thermo Fisher Scientific Triglycerides Reagent (#TR22421) and normalized per gram of liver tissue.
8
Liver Triglyceride Quantification
9
Tissue Triglyceride Quantification Protocol
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Tissue TAG levels were measured using Triglycerides Reagent (Thermo Fisher Scientific, TR22421) and Triglyceride Standard (Pointe Scientific, T7531STD) according to a modified protocol (53 (link)). In brief, tissue samples were homogenized in PBS (1/30, w/v). Then, 2.5 μL of the standard or sample homogenization and 250 μL of the reagent were added to each microplate well. After incubation at 37°C for 10 minutes, absorbances at 500 nm were measured using a plate reader from Tecan.
10
Triglyceride Quantification in Adipocytes
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Cells were plated at 5000/cm2 density, and adipogenesis was induced as described. Then cells were lysed using PBS + 1% triton X100. Triglyceride concentration was determined using Thermo Scientific™ Triglycerides Reagent according to the manufacturer’s protocol. A C2-C10 triglyceride mix was used to generate the standard curve. Total protein concentration was measured using Pierce 660 nm protein assay.
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