At the end of the study, mice were sacrificed. The blood was collected and plasma insulin, TNF-α, PAI-1, and resistin concentrations were measured using a mouse adipokine assay kit (Millipore). Plasma total cholesterol levels were measured by enzymatic colorimetric assay using a kit from Wako Chemicals. In addition, lipoprotein cholesterol distributions were evaluated in individual serum samples after fractionation by fast protein liquid chromatography (FPLC) gel filtration on a single Superose 6 column as described previously [13 (link)]. In brief, a Bio-Rad Biologic DuoFlow System with BioFrac Fraction Collector and Superose 6 10/300GL Column (GE Healthcare) was used. The elusion buffer contained 0.15 M NaCl and 1 mM EDTA. After loading of 50 μL plasma into the system, the flow rate should not exceed 0.5 ml/min. The fractionation was started at 10 ml and ended at 26 ml with each fraction size of 0.5ml. Total of 32 fractions were collected. Cholesterol concentrations from fractions 1–32 were further measured by using Cholesterol E Enzymatic Kit (WAKO).
Cholesterol e enzymatic kit
Manufactured by Fujifilm
The Cholesterol E Enzymatic Kit is a laboratory reagent used for the quantitative determination of cholesterol levels in biological samples. The kit employs an enzymatic method to measure the concentration of cholesterol in the sample.
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3 protocols using cholesterol e enzymatic kit
1
Lipoprotein Cholesterol Profiling in Mice
2
Cholesterol and Triglyceride Analysis by FPLC
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Total serum cholesterol (Cholesterol E Enzymatic Kit, Wako Pure Chemical Industries kits Mountain View, CA) and triglycerides concentrations (L-Type Triglyceride M Enzyme Color A and Color B, Wako Pure Chemical Industries kits Mountain View, CA) were analyzed. Lipoprotein cholesterol and triglyceride distributions were analyzed from individual serum samples (50 µl) fractioned by fast protein liquid chromatography (FPLC) (Leica CM1860). Fractions were collected and cholesterol and triglyceride concentrations were determined with the Wako kits described above. For each group, 4–5 samples around the mean total serum cholesterol concentration were analyzed.
3
Serum Lipid Profiling by FPLC
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After anesthesia, blood was collected by cardiac puncture and centrifuged to isolate serum. Total serum cholesterol (Cholesterol E Enzymatic Kit, Wako Pure Chemical Industries kits Mountain View, CA) and triglycerides concentrations (L-Type Triglyceride M Enzyme Color A and Color B, Wako Pure Chemical Industries kits Mountain View, CA) were measured with commercially available kits. Individual 50 µl serum samples were fractioned by fast protein liquid chromatography (FPLC) using a Superose 6 column. Cholesterol and triglyceride concentrations were measured in each fraction with the kits above. Lipoprotein distributions were analyzed in 4-5 samples selected to be around the mean total serum cholesterol concentration.
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