The blood was collected once a week before the end of the experiment and at 4 h intervals. All the plasma samples were run in duplicate in a single assay. The plasma levels of glucose (Glu), total cholesterol (TC), low-density triglycerides (TG), non-esterified fatty acids (NEFA), high-density lipoproteins, low-density lipoprotein, and apolipoproteins (A1, B, E) were individually measured at the Clinical Biochemistry Service of Nan Jing general hospital (Nanjing, China), using Beckman AU5811 analyzer (Beckman-Coulter, 250S.Kraemer Boulevard Brea, USA). The plasma melatonin level was determined using high performance liquid chromatography (HPLC, Thermo Ultimate 3000; Thermo Fisher Scientific, Waltham, MA, USA) as described previously by Yin et al [16 (link)]. Leptin and insulin levels were measured in plasma using commercial bovine ELISA kit (Shanghai Enzyme-linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions. For plasma insulin analysis, the results with intra-assay coefficient of variation (CV) of 3.0% to 6.0% were taken into consideration. For leptin analysis, results were acceptable with a CV of less than 5%.
Au5811 analyzer
Manufactured by Beckman Coulter
Sourced in China, United States
The AU5811 analyzer is a clinical chemistry analyzer designed for high-volume clinical laboratories. It is capable of performing a wide range of routine and specialty diagnostic tests on a variety of biological samples, including blood, urine, and other body fluids. The instrument uses advanced spectrophotometric technology to provide accurate and reliable results.
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4 protocols using au5811 analyzer
1
Comprehensive Metabolic Profiling Protocol
2
Plasma Metabolite and Hormone Analysis
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The concentrations of triglyceride (TG) and nonesterified fatty acids (NEFA) in plasma samples were determined by using Beckman AU5811 analyzer (Beckman-Coulter, Inc.250S.Kraemer Boulevard Brea, CA, USA) as previously reported [8 (link)]. A commercially available porcine specific ELISA kit (RD SYSTEMS, Minneapolis, MN, USA) and an Amplex Red hydrogen peroxide assay kit (Invitrogen, Carlsbad, CA, USA) were utilized to measure plasma leptin and hydrogen peroxide (H2O2) content, respectively, according to the manufacturer’s instructions as previously described [5 (link),17 (link)]. All blood samples were determined in duplicate in single assay. The minimum detection limit was 3.2 pg/dL, 0.1 pmol/L, 0.2 ng/mL, and 0.2 pmol/L for TG, NEFA, Leptin, and H2O2, respectively. For Leptin and H2O2 analysis, within-assay coefficient of variation (CV) was acceptable when less than 5%.
3
Serum Creatinine Measurement Protocol
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Serum creatinine was measured to assess renal function and analyzed by an AU5811 analyzer (Beckman Coulter) using the enzymatic assay method (DONGOU, Cat: 59400377071).
4
Biomarker Measurement for Metabolic Health
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C-reactive protein (CRP), triglycerides, total cholesterol, and high-density lipoprotein (HDL) cholesterol were measured using a Beckman Coulter AU5811 analyzer. The interassay coefficients of variations were as follows: CRP 6.5% at 0.9 mg/L and <2% at >2.5 mg/L; triglycerides: <2.1% at 1-2 mmol/L; cholesterol: <1.4% at 3.5-7 mmol/L; and HDL cholesterol: <1.4% at 1-2.2 mmol/L. Low-density lipoprotein (LDL) cholesterol was calculated, using the Friedewald formula (26) . Insulin was measured using an electrochemiluminescence immunoassay on the Cobas E411 (Roche Diagnostics GmbH); the lower limit of detection was 2 mE/ L, and the interassay variation was <4.4%.
Adiponectin was determined in a Quantikine ELISA (Acrp30; R&D Systems); the lower limit of detection was 3.9 ng/mL for undiluted samples. Plasma samples were diluted 500-fold, and the interassay variation coefficients were <6.5% at 30-175 ng/mL (n ¼ 9 undiluted control samples).
Leptin was determined using the Human Leptin radioimmunoassay #HL-81HK (EMD Millipore); the lower limit of detection was 0.7 ng/mL. The interassay coefficients of variation were <6% at 4-60 mg/L (n ¼ 7 undiluted control samples).
Adiponectin was determined in a Quantikine ELISA (Acrp30; R&D Systems); the lower limit of detection was 3.9 ng/mL for undiluted samples. Plasma samples were diluted 500-fold, and the interassay variation coefficients were <6.5% at 30-175 ng/mL (n ¼ 9 undiluted control samples).
Leptin was determined using the Human Leptin radioimmunoassay #HL-81HK (EMD Millipore); the lower limit of detection was 0.7 ng/mL. The interassay coefficients of variation were <6% at 4-60 mg/L (n ¼ 7 undiluted control samples).
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