Insulin, glucagon, glycogen, and TAG were quantified using Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem, 90080), Mouse Glucagon ELISA Kit (Crystal Chem, 81518), Glycogen Assay Kit (BioVision, K646-100), and Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma, MAK266), following manufacturers’ instructions.
Triglyceride quantification colorimetric fluorometric kit
Manufactured by Merck Group
Sourced in United States
The Triglyceride Quantification Colorimetric/Fluorometric Kit is a laboratory tool designed for the quantitative determination of triglyceride levels. It utilizes a colorimetric or fluorometric method to measure the concentration of triglycerides in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Ultra sensitive mouse insulin elisa kit, by Crystal Chem (2 mentions)
Free fatty acid quantitation kit, by Merck Group (2 mentions)
Glycogen assay kit, by Abcam (1 mentions)
Mouse glucagon elisa kit, by Crystal Chem (1 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (1 mentions)
Glycogen colorimetric fluorometric assay kit, by Abcam (1 mentions)
Spotchem ez sp 4430, by Arkray (1 mentions)
Mouse ultrasensitive insulin elisa kit, by Crystal Chem (1 mentions)
Prism v9, by GraphPad (1 mentions)
9 protocols using triglyceride quantification colorimetric fluorometric kit
1
Quantitative Analysis of Key Metabolites
2
Metabolic Profiling in Mice
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Blood glucose was measured via the tail using the Accu-Chek Aviva Plus Blood Glucose Monitoring System. Blood serum was collected after centrifugation at 3000 rpm for 10 min at 4°C and stored at −80°C. The following commercially available kits were used to measure metabolic parameters in the serum, according to the manufacturer’s instructions: triglycerides, Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma-Aldrich, MAK266-1KT); free fatty acids, Free Fatty Acid Quantitation Kit (Sigma-Aldrich, MAK044-1KT); insulin, Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem, 90080).
3
Quantifying Liver Triglycerides
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Per 100 mg of flash-frozen liver, 1 mL of 5% NP-40 lysis buffer was added to generate a homogenate. The homogenized tissue samples were then subject to two cycles of heating at 80–100°C for 5 minutes followed by cooling at room temperature. The resulting mixture was centrifugated, and supernatant was collected. Triglyceride concentration of the supernatant was measured using Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma, MAK266) following manufacturer instructions.
4
Measurement of cellular triglyceride levels
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Cellular triglyceride levels were measured on cells plated in 96-well plates, using the Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma-Aldrich) according to the manufacturer’s instructions.
For imaging, cells transfected with either siC or siCKB were fixed in 4% PFA for 15 min at room temperature and washed twice with PBS. Cells were stained with BODIPY 493/503 (diluted 1:2,500, ThermoFisher) to stain accumulated lipids and Hoechst (diluted 1:5,000) for 15 min. Cells were then washed four times with PBS and images were acquired using CREST V3 confocal system (Crest Optics) mounted on an inverted Nikon Ti2 microscope equipped with a Prime BSIexpress sCMOS camera (pixel size 6.5 μm) from Photometrics. A Nikon ×60/1.4 oil objective was used to acquire images.
For imaging, cells transfected with either siC or siCKB were fixed in 4% PFA for 15 min at room temperature and washed twice with PBS. Cells were stained with BODIPY 493/503 (diluted 1:2,500, ThermoFisher) to stain accumulated lipids and Hoechst (diluted 1:5,000) for 15 min. Cells were then washed four times with PBS and images were acquired using CREST V3 confocal system (Crest Optics) mounted on an inverted Nikon Ti2 microscope equipped with a Prime BSIexpress sCMOS camera (pixel size 6.5 μm) from Photometrics. A Nikon ×60/1.4 oil objective was used to acquire images.
5
Colorimetric Triglyceride Quantification
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Triglyceride content was determined in both HepG2 and human subcutaneous pre-adipocyte cells using the Triglyceride Quantification Colorimetric/Fluorometric Kit from Sigma, according to the manufacturer’s protocols. TG is converted to free fatty acids and glycerol. The glycerol is then oxidized to generate a colorimetric (570 nm)/fluorometric (λex = 535 nm/λem = 587 nm) product.
6
Adipocyte Metabolic Analysis in Lamb
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Lamb adipocytes in the different treatment groups were assayed for glycerol phosphate dehydrogenase (GPDH) content, triglyceride content, and FFA content using the GPDH Activity Assay Kit (MAK208, Sigma-Aldrich, St. Louis, MO, USA), Triglyceride Quantification Colorimetric/Fluorometric Kit (MAK266, Sigma-Aldrich, St. Louis, MO, USA), and Free Fatty Acid Quantitation Kit (MAK044, Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer’s protocols.
7
Triglyceride Quantification in Cultured Cells
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Lipids were extracted and dissolved in chloroform. An aliquot (30 μL) was removed
from each sample for quantification. Cultured cells were directly lysed in 1%
Triton X-100 in PBS. After centrifugation at 500 g for 10 min
at 4°C, a 30-μL aliquot was taken from each sample for measurement. TG were
measured using Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma,
mak266), according to the manufacturer's instructions.
from each sample for quantification. Cultured cells were directly lysed in 1%
Triton X-100 in PBS. After centrifugation at 500 g for 10 min
at 4°C, a 30-μL aliquot was taken from each sample for measurement. TG were
measured using Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma,
mak266), according to the manufacturer's instructions.
8
Biochemical Analyses of Liver Function
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The measurements of ALT, AST, total cholesterol, HDL, triglycerides, and bilirubin level in serum were performed using biochemical analyzer SPOTCHEM EZ SP-4430 (ARKRAY, Kyoto, Japan) according to the vendor’s instructions. Triglyceride content in the liver was assessed by Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma-Aldrich, St. Louis, MO, USA), whereas glycogen level was analyzed using Glycogen Colorimetric/Fluorometric Assay Kit (BioVision, Milpitas, CA, USA). The urinary uric acid concentration was tested according to the vendor’s instruction (CORMAY, Warsaw, Poland).
9
Quantifying Metabolic Biomarkers in ob/ob Mice
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Fasting insulin and triglycerides from blood collected at euthanasia were quantified using a mouse ultrasensitive insulin ELISA kit (Crystal Chem, Elk Grove Village, IL) and a Triglyceride Quantification Colorimetric/Fluorometric Kit (Sigma).
2.9 Histology. Histological analysis of pancreata was conducted per established methods, methodological details can be found in Supplementary Information.
2.10 Genotyping. Formalin fixed liver tissue was used to genotype each animal to confirm the presence or absence of the ob/ob mutation. Samples were sent to Transnetyx (Cordova, TN) and results can be found in Supplementary Information.
2.11 Statistics. Data were analyzed using GraphPad Prism v9.1.2 (La Jolla, CA). When values were missing, a mixed-effects model was used. When initial one-way or two-way ANOVA indicated a significant overall treatment effect or interaction (P<0.05), individual means were compared using Tukey's or Sidak's post-hoc tests to control for multiple comparisons (α = 0.05).
2.9 Histology. Histological analysis of pancreata was conducted per established methods, methodological details can be found in Supplementary Information.
2.10 Genotyping. Formalin fixed liver tissue was used to genotype each animal to confirm the presence or absence of the ob/ob mutation. Samples were sent to Transnetyx (Cordova, TN) and results can be found in Supplementary Information.
2.11 Statistics. Data were analyzed using GraphPad Prism v9.1.2 (La Jolla, CA). When values were missing, a mixed-effects model was used. When initial one-way or two-way ANOVA indicated a significant overall treatment effect or interaction (P<0.05), individual means were compared using Tukey's or Sidak's post-hoc tests to control for multiple comparisons (α = 0.05).
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