Intracellular TG content was measured by enzymatic methods using the InfinityTM Triglycerides Liquid Stable Reagent (Thermo Scientific), as previously described [71 (link)]. Briefly, BAT samples were homogenized and diluted in saline at a final concentration of 50 mg/mL. BAT homogenates were diluted (1:1) in 1% deoxycholate (Sigma) and incubated at 37 °C for 5 min. Afterwards, samples were diluted 1:100 in the Infinity™ Triglycerides Liquid Stable Reagent (Thermo Scientific) and incubated for 30 min at 37 °C. The resulting dye was measured based on its absorbance at 550 nm. Concentrations were determined compared with a standard curve of triglycerides (Infinity™ Triglycerides Standard, Thermo Scientific). The protein content of the preparations was measured by the Bradford assay. All assays were performed in duplicate.
Infinity triglycerides standard
Manufactured by Thermo Fisher Scientific
The Infinity™ Triglycerides Standard is a reference material used for the quantitative determination of triglycerides in clinical laboratory testing. It provides a standardized solution with a known concentration of triglycerides to calibrate and verify the performance of triglycerides assays.
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6 protocols using infinity triglycerides standard
1
Measuring Intracellular Triglyceride Content
2
Triglyceride Quantification in Tissue Homogenates
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Tissue were homogenized and diluted in saline at a final concentration of 50 mg/mL as previously described61 (link), 65 (link). Homogenates were diluted (1:1) in 1% deoxycholate (Sigma-Aldrich) and incubated at 37 °C for 5 min. For triglyceride measurements, samples were diluted 1:100 in the reagent (Infinity™ Triglycerides Liquid Stable Reagent, Thermo Fisher Scientific) and incubated for 30 min at 37 °C. The resulting dye was measured based on its absorbance at 550 nm with a Sunrise ELISA plate reader (Tecan, Männedorf, Switzerland). Concentrations were determined compared with a standard curve of triglycerides (Infinity™ Triglycerides Standard, Thermo Fisher Scientific). The protein content of the preparations was measured by the Bradford method, using BSA (Sigma-Aldrich) as standard. All assays were performed in duplicate.
3
Pancreatic Triglyceride Quantification Protocol
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Pancreatic TG content was measured by enzymatic methods using the Infinity™ Triglycerides Liquid Stable Reagent (Thermo Scientific), as previously described (13 (link)). Briefly, pancreas samples were homogenized and diluted in saline at a final concentration of 50 mg/mL. Pancreas homogenates were diluted (1:1) in 1% deoxycholate (Sigma) and incubated at 37°C for 5 min. Afterwards, samples were diluted 1:100 in the Infinity™ Triglycerides Liquid Stable Reagent (Thermo Scientific) and incubated for 30 min at 37°C. The resulting dye was measured based on its absorbance at 550 nm. Concentrations were determined compared with a standard curve of TG (Infinity™ Triglycerides Standard, Thermo Scientific). The protein content of the preparations was measured by the Bradford assay. All assays were performed in duplicate.
4
Intracellular Triglyceride Quantification
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Intracellular TG content was measured by enzymatic methods using the Infinity™ Triglycerides Liquid Stable Reagent (TR22421, Thermo Scientific), according to manufacturer's protocol. Concentrations were determined compared with a standard curve of triglycerides (TR23923, Infinity™ Triglycerides Standard, Thermo Scientific). The protein content of the preparations was measured by the Bradford assay.
5
Hepatic Triglyceride Quantification Protocol
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The hepatic triglyceride content was measured by enzymatic methods, in accordance with previously published procedures [22] (link). Briefly, tissues were homogenized and diluted in saline at a final concentration of 50 mg/mL. Homogenates were diluted (1∶1) in 1% deoxycholate (Sigma) and incubated at 37 °C for 5 min. For triglyceride measurements, samples were diluted 1∶100 in the reagent (Infinity Triglycerides Liquid Stable Reagent, Thermo Electron) and incubated for 30 min at 37 °C. The resulting dye was measured based on its absorbance at 550 nm with a Sunrise ELISA plate reader (Tecan). Concentrations were determined compared with a standard curve of triglycerides (Infinity Triglycerides Standard, Thermo Electron). The protein content of the preparations was measured by the Bradford method, using BSA (Sigma) as standard. All assays were performed in duplicate.
6
Quantification of Intrahepatic Triglycerides
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Blood assays were determined as previously described26 (link). Intrahepatic TG content was measured by enzymatic methods, in accordance with previously published procedures12 (link). Briefly, liver biopsies were homogenized and diluted in saline at a final concentration of 50 mg/mL. Homogenates were diluted (1:1) in 1% deoxycholate (Sigma, St. Louis, MO, USA) and incubated at 37 °C for 5 min. For TG measurements, samples were diluted 1:100 in the reagent (Infinity™ Triglycerides Liquid Stable Reagent, Thermo Electron Corporation, Melbourne, Australia) and incubated for 30 min at 37 °C. The resulting dye was measured based on its absorbance at 550 nm. Concentrations were determined compared with a standard TG curve (Infinity™ Triglycerides Standard, Thermo Electron Corporation). The protein content of the preparations was measured by the Bradford method, using bovine serum albumin (BSA) (Sigma) as standard. All assays were performed in duplicate.
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