After treating flies on RD and HFD for 7 days, a group of males (n = 7) and females (n = 5) flies were separately weighed and transferred to 2ml, 2.8mm ceramic bead tubes (MoBio). More males were used because of their smaller size. Each group was done in triplicates. Flies were then homogenized in 0.05% Tween using the Precelly's 24 homogenizer (bertin technologies), following which 500ul supernatant was transferred to an eppy tube. The eppy tubes were heated at 70° for 5 min (to inactive lipases) with gentle shaking, centrifuged for 3 min, 13.2k rpm, and only the supernatants were used for both TG and protein assays. TG levels were measured using the Infinity TG kit (Cat#. TR22421, Thermo Fisher Scientific) and protein levels were measured using normal BCA protein assay (Sigma–Aldrich, Saint Louis, MO).
Infinity tg kit
Manufactured by Thermo Fisher Scientific
The Infinity TG kit is a laboratory diagnostic tool used for the quantitative determination of triglycerides in human serum or plasma. The kit utilizes an enzymatic colorimetric method to measure the concentration of triglycerides.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Precellys 24 homogenizer, by Bertin Technologies (1 mentions)
Complete mini edta free protease inhibitor, by Merck Group (1 mentions)
Nefa kit, by Fujifilm (1 mentions)
Total cholesterol e kit, by Fujifilm (1 mentions)
Elisa kit, by Mercodia (1 mentions)
Oxymax, by Columbus Instruments (1 mentions)
4 protocols using infinity tg kit
1
Measuring Triglycerides and Protein in Flies
2
Inhibition of Lipoprotein Lipase and Plasma Lipid Dynamics
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Mice were fasted for 4 h and injected intravenously with 1000 mg/kg body weight with the lipoprotein lipase inhibitor, Poloxamer-407. Tail vein blood was collected at t = 0, 1, 2, and 4 h. Plasma was supplemented with complete mini EDTA-free protease inhibitor (Sigma Aldrich) and snap frozen. Plasma TG was measured using Infinity TG kit (Thermo Scientific). ApoB-100 protein levels were measured by immunoblotting, as described earlier. At 1 h, the sample was diluted 1:5. At 2 and 4 h, they were diluted 1:10. 1.5 μl of plasma (or 1.5 μl of diluted plasma) was heated at 95°C for 5 min in 2× denaturing sample buffer. Plasma samples from each time point were run on the same gel. Equal loading was confirmed by visualization of albumin with Ponceau staining. To compare between gels, a sample from each time point was run on the t = 0 gel (Fig. S3 A).
3
Protein and Triglyceride Quantification
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Protein concentrations were assayed with a BCA protein assay kit (Thermo Scientific cat# 23227). TG concentrations were assayed with the Infinity TG kit (Thermo Scientific cat# TR2241).
4
Metabolic Phenotyping of Mice on Diets
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We performed intraperitoneal glucose tolerance tests (ipGTT) by injecting glucose (2g/kg for NCD-fed mice, or 1g/kg for HFD fed mice) after an overnight fast. Insulin tolerance tests were performed by injecting insulin (0.75 units/kg) after a 4-hr fast, and intraperitoneal pyruvate tolerance tests by injecting sodium pyruvate (2g/kg) after an overnight fast (17) . We measured insulin levels with an ELISA kit (Mercodia) and glucagon levels with radioimmunoassay kit (MilliporeSigma). We estimated body composition by nuclear magnetic resonance (Bruker Optics). We measured food intake, energy expenditure, and respiratory exchange rate (RER) with a Comprehensive Laboratory Animal Monitoring System (CLAMS, Oxymax) (Columbus Instruments, OH, USA). Plasma triglyceride (TG), total cholesterol E and non-esterified fatty acid (NEFA) were measured with commercially available kits following manufacturers' instructions: Infinity TG kit (Thermo Fisher Scientific), total cholesterol E kit (Wako Diagnostics), and NEFA kit (Wako Diagnostics).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 8, 2020. ; https://doi.org/10.1101/2020.10.06.328963 doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted October 8, 2020. ; https://doi.org/10.1101/2020.10.06.328963 doi: bioRxiv preprint
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