Plasma cholesterol and triglycerides were determined by CHOD-PAP kit (Roche/Hitachi) and GPO-PAP kit (Roche/Hitachi) respectively, according to the manufacturer’s instructions.
Chod pap kit
Manufactured by Roche
Sourced in United States
The CHOD-PAP kit is a diagnostic reagent kit used for the quantitative determination of cholesterol in serum and plasma. It employs the CHOD-PAP method, which involves the enzymatic hydrolysis and oxidation of cholesterol esters to cholesterol, which is then measured colorimetrically.
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5 protocols using chod pap kit
1
Plasma Cholesterol and Triglyceride Analysis
2
Liver Lipid Profiling Protocol
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Liver lipids were extracted according to Bligh and Dyer
[33 (link)], solvents were evaporated under nitrogen and the samples were re-dissolved in isopropanol before analysis. Lipids from liver extracts or plasma were then measured enzymatically on a Hitachi 917 system (Roche Diagnostics, Mannheim, Germany) using kits for analyzing total TAG (GPO-PAP kit, Roche Diagnostics), cholesterol (CHOD-PAP kit, Roche Diagnostics), total PLs (bioMérieux SA, Marcy l'Etoile, France) and NEFA (FS kit, DiaSyS, Holzheim, Germany). Aliquots of extracted liver lipids were separated by thin layer chromatography using silica gel plates (Merck, Darmstadt, Germany) and hexane:diethylether (1:1) as the liquid phase. The absolute levels of fatty acids of the diets, plasma and the TAG and PL fractions from livers were analyzed using gas chromatography as described previously by Grimstad et al.
[31 (link)]. Lipoproteins were analyzed by size exclusion chromatography of plasma samples from individual mice (five mice in each group) according to Parini et al.
[34 (link)].
[33 (link)], solvents were evaporated under nitrogen and the samples were re-dissolved in isopropanol before analysis. Lipids from liver extracts or plasma were then measured enzymatically on a Hitachi 917 system (Roche Diagnostics, Mannheim, Germany) using kits for analyzing total TAG (GPO-PAP kit, Roche Diagnostics), cholesterol (CHOD-PAP kit, Roche Diagnostics), total PLs (bioMérieux SA, Marcy l'Etoile, France) and NEFA (FS kit, DiaSyS, Holzheim, Germany). Aliquots of extracted liver lipids were separated by thin layer chromatography using silica gel plates (Merck, Darmstadt, Germany) and hexane:diethylether (1:1) as the liquid phase. The absolute levels of fatty acids of the diets, plasma and the TAG and PL fractions from livers were analyzed using gas chromatography as described previously by Grimstad et al.
[31 (link)]. Lipoproteins were analyzed by size exclusion chromatography of plasma samples from individual mice (five mice in each group) according to Parini et al.
[34 (link)].
3
Lipoprotein Profile Analysis in Mice
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Plasma total cholesterol levels, triglyceride levels and pooled lipoprotein profiles were determined at 0, 4, 8, 12, and 16 weeks. Total cholesterol and triglyceride concentrations were measured using the CHOD-PAP and TG GPO-PAP analyzer kits, respectively (Roche/Hitachi, Roche Diagnostics, Rotkreuz, Switzerland). Pooled lipoprotein profiles were assessed by fast protein liquid chromatography (ÄKTA). Cholesterol and phospholipid profiles in the pooled samples were measured using the CHOD-PAP kit (Roche/Hitachi, Roche Diagnostics, Rotkreuz, Switzerland) and the Phospholipids Assay Kit (INstruchemie, Delfzijl, Netherlands), respectively. Liver free cholesterol, cholesterol esters, and triglycerides levels were determined in eight mice per group from the 30 µL/mouse/day groups as described previously (Havekes et al., 1987 (link)).
4
Quantifying Adipokine Secretion in ASCs
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Conditioned media from induced ASCs and 3T3-L1 wells, as well as non-induced controls were collected at 14-days post-induction, where the media had been conditioned for 48 hours prior to analysis. Leptin and adiponectin content in the media was measured by ELISA following the manufacturer’s protocols (Crystal Chem Inc., IL, USA). Total leptin and adiponectin levels in the supernatants were normalized to total intracellular protein content measured using the Bio-Rad Protein Assay. Additionally, for the in vivo studies, ELISAs (ALPCO, NH, USA) were performed following the manufacturer’s protocols for insulin and adiponectin, while cholesterol was assessed by CHOD-PAP kit (Roche Diagnostics, Indianapolis, IN) and triglyceride analysis was conducted by Triglycerol/Glycerol kit (Roche Diagnostics, Indianapolis, IN) following manufacturer’s protocols. Analyses for the in vivo studies were conducted using serum collected from congenic mice fed on a 5-week HFD.
5
Glucose Tolerance Test in Mice
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Mice were fasted 4 hours prior to testing. Fasted blood glucose was measured via a glucometer (OneTouch Ultra). Glucose tolerance testing was conducted by administration of 1 g/kg of glucose by intraperitoneal injection, and blood glucose was monitored at 0, 15, 30, 60, and 120 minutes via tail vein puncture. Glucose area under the curve (AUC) was calculated. At sacrifice, blood was collected, and plasma was isolated. ELISAs (ALPCO, NH, USA) were performed following the manufacturer's protocol for insulin, cholesterol was assessed by CHOD-PAP kit (Roche Diagnostics, Indianapolis, IN), and triglyceride analysis was conducted by Triglycerol/Glycerol kit (Roche Diagnostics, Indianapolis, IN) following manufacturer's protocols.
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