Total plasma cholesterol and triglyceride concentrations were measured in a microtiter assay, using Infinity™ commercial kits (Thermo Scientific, Madrid, Spain), glucose (BioSystems, Barcelona, Spain) and non-esterified fatty acids (NEFA) (Fujifilm Wako chemicals, Richmond VA, USA) according to the manufacturer’s instructions. Total serum apolipoprotein A1 (APOA1) was quantified by ELISA (34 (link)) and arylesterase activity of paraoxonase (PON1) as previously described (35 (link)). Plasma lipoprotein profile was determined in 100 μl of pooled plasma samples from each group and sex by fast protein liquid chromatography (FPLC) gel filtration using a Superose 6B column (GE Healthcare, Chicago, Il, USA) as previously described (36 (link)). The presence of reactive oxygen species (ROS) was assessed by measuring the conversion of 2,7- dichlorofluorescein diacetate into fluorescent dichlorofluorescein (37 (link)) in FPLC-isolated fractions corresponding to the different lipoproteins (38 (link)), using equal amount of total cholesterol for each fraction.
Superose 6b column
Manufactured by GE Healthcare
Sourced in United States
The Superose 6B column is a gel filtration chromatography column used for the size-based separation of proteins and other macromolecules. It is designed to provide efficient and high-resolution separation of molecules with a molecular weight range of 5,000 to 500,000 Daltons.
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Lab products found in correlation
4 protocols using superose 6b column
1
Comprehensive Lipid and Oxidative Profiling
2
Comprehensive Lipoprotein and Metabolite Analysis
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Total plasma cholesterol and triglyceride concentrations were measured in a microtiter assay, using InfinityTM commercial kits (Thermo Scientific, Madrid, Spain), glucose (BioSystems, Barcelona, Spain) and non-esterified fatty acids (NEFA) (Fujifilm Wako chemicals, Richmond VA, USA) according to the manufacturer’s instructions. Total serum apolipoprotein A1 (APOA1) was quantified by ELISA [25 (link)] and arylesterase activity of paraoxonase (PON1) as previously described [26 (link)]. Plasma lipoprotein profile was determined in 100 μL of pooled plasma samples from each group and sexes by fast protein liquid chromatography (FPLC) gel filtration using a Superose 6B column (GE Healthcare, Chicago, Il, USA) in 48 fractions as previously described [27 (link)].
3
Plasma Lipid and Lipoprotein Analysis
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Total plasma cholesterol and triglyceride concentrations were measured in a microtiter assay using commercial Infin-ity™ kits (Thermo Scientific, Madrid, Spain), glucose (Bio-Systems, Barcelona, Spain) and HDL cholesterol (HDL-c) according to the Grove protocol [16] . Total serum apolipoprotein A1 (APOA1) and apolipoprotein A4 (APOA4) were quantified by ELISA as previously described [33] (link). Plasma lipoprotein profile was determined in 100 µL of pooled plasma samples from each group and sex by fast protein liquid chromatography (FPLC) gel filtration using a Superose 6B column (GE Healthcare, Chicago, Il, USA) as previously described [29] (link).
4
Plasma Lipid and Oxidative Profiling
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After the experimental period, animals were sacrificed by suffocation with CO2, blood (0, 7–1 ml per mouse) was collected via cardiac puncture and centrifuged at 3000 rpm for 5 minutes at 4°C to obtain plasma. Total plasma cholesterol and triglyceride concentrations were measured in a microtitre assay, using Infinity commercial kits (Thermo Scientific Madrid, Spain). Plasma HDL-cholesterol was quantified in the supernatant after precipitation of apoB particles with phosphotungstic acid–MgCl2 (Roche, Barcelona, Spain). Glucose and non-esterified fatty acids were determined using kits from BioSystems (Barcelona, Spain) and Wako (Madrid, Spain). Paraoxonase was assayed as arylesterase activity by the rate of phenylacetate hydrolysis, as described previously [60] (link). APOA1 and APOA4 were quantified by ELISA using specific polyclonal antibodies (Biodesign and Santa Cruz Biotechnology), as described previously [61] (link). Malondialdehyde was assayed following Conti's spectrofluorimetric method [62] (link). Plasma lipoprotein profile was determined in 100 µl of pooled plasma samples from each group by fast protein liquid chromatography (FPLC) gel filtration [63] (link) using a Superose 6B column (GE Healthcare), and the cholesterol, phosphatidylcholine and sphingomyelin contents in each fraction were measured as described [49] (link).
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