Blood was collected on the first day and last day of each intervention period after a 12–hour fast by trained research staff. Specimens were locally processed and stored at −80°C until analyses. Glucose was measured on a Roche Module P chemistry autoanalyzer (Roche Diagnostic Inc., Indianapolis, IN) at the Northwest Lipid Research Laboratories (University of Washington, WA). Insulin was measured using a Tosoh 2000 autoanalyzer (Tosoh Biosciences Inc., South San Francisco, CA) at the Diabetes Endocrinology Research Center Immunoassay Laboratory (University of Washington, WA). The rest of the biomarkers assessments and the genotyping were conducted at the FHCRC Biomarker Core Laboratory and the Molecular Epidemiology Laboratories. Immunoassays were used to measured total adiponectin (Total Adiponectin EIA, Aplco), IGF-1 (Human IGF-I Quantikine ELISA, R&D Systems), IGFBP-3 (Human IGFBP-3 Quantikine ELISA, R&D Systems), and IL-6 (Human IL-6 Quantikine HS ELISA, R&D Systems). CRP was measured using CRP (3 (link))-Wide Range reagent (Kamiya Biomedical Company) on Roche Cobas Mira chemistry analyzer with a high sensitivity protocol. The intra-assay CVs were 0.7%, 7.8%, 1.3%, 1.5%, 1.8%, 2.3%, and 3.3% for glucose, insulin, adiponectin, IGF-1, IGFBP-3, IL-6, and CRP, respectively. The details of specimen collection and analysis have been previously described (14 (link)).
Human il 6 quantikine hs elisa
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Human IL-6 Quantikine HS ELISA is a quantitative sandwich enzyme immunoassay designed to measure human interleukin-6 (IL-6) levels in cell culture supernates, serum, and plasma. It is a high-sensitivity assay with a minimum detectable dose typically less than 0.16 pg/mL.
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Lab products found in correlation
Human igfbp 3 quantikine elisa, by R&D Systems (2 mentions)
Module p, by Roche (2 mentions)
Human igf 1 quantikine elisa, by R&D Systems (2 mentions)
2000 autoanalyzer, by Tosoh (2 mentions)
Cobas mira chemistry analyzer, by Roche (2 mentions)
Human total adiponectin acrp30 quantikine elisa kit, by R&D Systems (1 mentions)
Advia 1650 chemistry system analyser, by Siemens (1 mentions)
Cobas c 311 analyzer, by Hitachi (1 mentions)
Human il 18 elisa, by Medical & Biological Laboratories (1 mentions)
Human chemerin elisa, by BioVendor (1 mentions)
Varioskan lux multimode microplate reader, by Thermo Fisher Scientific (1 mentions)
Human tnf α quantikine hs elisa, by R&D Systems (1 mentions)
Human leptin elisa kit, by Merck Group (1 mentions)
10 protocols using human il 6 quantikine hs elisa
1
Biomarker Measurement and Analysis
2
Cardiometabolic Risk Markers Measurement
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Participants underwent a medical examination that included measurement of systolic blood pressure (SBP) and diastolic blood pressure (DBP), and obtaining a fasting blood sampling [50 (link)]. We retrieved, processed, and measured more cardiometabolic risk markers from fasting blood samples of participants who fulfilled the inclusion in the trajectory analysis. Serum cholesterol (total, LDL-C and HDL-C) were measured at the Paediatric Clinic Dortmund with the Advia 1650-Chemistry System analyser (Siemens Healthcare Diagnostics, Eschborn, Germany). We measured fasting plasma glucose (FPG) on a Roche/Hitachi Cobas c 311 analyzer (Basel, Switzerland). Triglycerides and high-sensitivity C-reactive protein (CRP) with the Roche/Hitachi Cobas c311 analyser (Roche diagnostics, Mannheim, Germany), interleukin (IL)-6 with the Human IL-6 Quantikine HS ELISA (R&D Systems, Wiesbaden, Germany), IL-18 with the Human IL‐18 ELISA (Medical and Biological Laboratories, Nagoya, Japan), adiponectin with the Human Total Adiponectin/Acrp30 Quantikine ELISA kit (R&D Systems), chemerin with the Human Chemerin ELISA (BioVendor, Brno, Czech Republic), and leptin with leptin Quantikine ELISA (R&D System) as described [51 (link), 52 (link)]. We considered SBP, DBP, triglyceride, HDL-c, FPG, CRP, IL‐6, IL‐18, adiponectin, chemerin, and leptin for the current study.
3
Measuring Inflammatory Markers in Serum
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In parallel with cfDNA, IL-6, and PGF2α levels were measured to confirm the inflammatory response induced by exercise. IL-6 was measured using Human IL-6 Quantikine HS ELISA (Cat.HS600C; R&D Systems, Minneapolis, MN, USA). The assay was performed according to the product protocol, and 100 μl of serum sample was used without dilution. The absorbance was measured at 450 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). A 4PL curve was generated from the absorbance of the standard solution using ImageJ, and the level of IL-6 in each serum sample was calculated. PGF2α was measured using a Prostaglandin F2 alpha ELISA Kit (Cat.KA0310; Abnova, Taipei, Taiwan). The manufacturer's instructions were followed, and 100 μl of a 10-fold diluted serum sample was used. The absorbance was measured at 405 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). The binding rates of the standard solution and each serum sample were calculated, and a 4PL curve was generated from the binding rates of the standard solution using ImageJ. The PGF2α concentration in each serum sample was calculated.
4
Biomarker Measurement and Analysis
5
Metabolic and Adipokine Profiles
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Fasting blood samples were collected on the morning of surgery after a 12h-overnight fast. From these samples, cholesterol and triglyceride levels in both plasma and lipoprotein fractions were measured (21) .
The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin levels as previously described (22) . Enzyme-linked immunosorbent assay was performed on these samples for the following adipokines: leptin (Human Leptin ELISA kit; EMD Millipore; Billerica, MA, USA), adiponectin (Human Adiponectin ELISA Kit; B-Bridge International Inc; Santa Clara, CA, USA), interleukin-6 (IL-6) (Human IL-6 Quantikine HS ELISA; R&D Systems; Minneapolis, MN, USA) and tumor necrosis factor-α (TNF-α) (Human TNF-α Quantikine HS ELISA; R&D Systems; Minneapolis, MN, USA).
The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated from fasting glucose and insulin levels as previously described (22) . Enzyme-linked immunosorbent assay was performed on these samples for the following adipokines: leptin (Human Leptin ELISA kit; EMD Millipore; Billerica, MA, USA), adiponectin (Human Adiponectin ELISA Kit; B-Bridge International Inc; Santa Clara, CA, USA), interleukin-6 (IL-6) (Human IL-6 Quantikine HS ELISA; R&D Systems; Minneapolis, MN, USA) and tumor necrosis factor-α (TNF-α) (Human TNF-α Quantikine HS ELISA; R&D Systems; Minneapolis, MN, USA).
6
Influenza Antibody and Cytokine Assays
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Serum was collected at baseline and follow-up sessions in 9 ml vacutainers which were stored at room temperature until centrifugation (4000 rpm for 15 min at 4 °C), followed by storage at −80 °C until for later assessment. Anti-influenza antibody titres were determined using hemagglutination inhibition (HI) as described previously (Kok et al., 2011 (link)). Paired pre- and post-immunisation samples for each individual were tested in parallel against the antigens influenza A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2) and B/Brisbane/60/2008. The HI titre is the reciprocal of the highest dilution of serum that completely inhibited agglutination of red blood cells.
Plasma interleukin-6 (IL-6) measurement used high-sensitivity ELISA (Quantikine HS Human IL-6 ELISA, R&D Systems) according to manufacturer’s instructions. The reported sensitivity of the assay was 0.039 pg/mL, with recorded intra-assay and inter-assay variation both <10%.
Plasma interleukin-6 (IL-6) measurement used high-sensitivity ELISA (Quantikine HS Human IL-6 ELISA, R&D Systems) according to manufacturer’s instructions. The reported sensitivity of the assay was 0.039 pg/mL, with recorded intra-assay and inter-assay variation both <10%.
7
Plasma IL-6 Measurement Protocol
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Blood (6 ml) was collected into a vacutainer containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant (Becton Dickinson Diagnostics, Oxford, United Kingdom).
Samples were immediately centrifuged at 1500 g for 10 min at 4 °C and plasma was aliquoted and stored at -80 °C for later cytokine assessment of plasma interleukin-6 (IL-6).
Plasma IL-6 was assessed in duplicate using high-sensitivity enzyme-linked immunosorbent assay (ELISA; Quantikine HS Human IL-6 ELISA, R&D Systems, UK) in accordance with the manufacturer's instructions. The limit of detection of this assay was 0.11 pg/mL, with an intraassay coefficient of variation of 4.2%. All samples from the same participant were assayed in the same run. Assessment of IL-6 primarily acted as an inflammation check and no causal assumptions about the role of IL-6 in the observed effects can be made.
Samples were immediately centrifuged at 1500 g for 10 min at 4 °C and plasma was aliquoted and stored at -80 °C for later cytokine assessment of plasma interleukin-6 (IL-6).
Plasma IL-6 was assessed in duplicate using high-sensitivity enzyme-linked immunosorbent assay (ELISA; Quantikine HS Human IL-6 ELISA, R&D Systems, UK) in accordance with the manufacturer's instructions. The limit of detection of this assay was 0.11 pg/mL, with an intraassay coefficient of variation of 4.2%. All samples from the same participant were assayed in the same run. Assessment of IL-6 primarily acted as an inflammation check and no causal assumptions about the role of IL-6 in the observed effects can be made.
8
Fasting Blood Sampling for IL-6 and CRP
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Morning fasting blood samples were collected between 7:30-10:00 am via peripheral (primarily antecubital) venipuncture in an independent session closely following the FU2 assessment (typically within two weeks) from consenting participants (n=791). Samples were not obtained from any participants reporting acute illness or injury. Serum was collected in silicon-coated tubes with no additives and left at room temperature for 40 -60 minutes to allow for clot formation.
Samples were centrifuged (1300 g) for 20 minutes and then immediately transferred into 2 mL tubes and stored at -80º C until the day of the assay.
IL-6 and CRP were assayed in duplicate using commercially available enzyme-linked immunosorbent assays (IL-6: Quantikine HS Human IL-6 ELISA, R&D Systems, Minneapolis, MN, USA; CRP: EIA-3954 High Sensitivity C-Reactive Protein ELISA DRG International Inc., USA). Intra-(IL-6: 5%, CRP: 4%) and inter-(IL-6: 14%, CRP: 13%) assay coefficients of variation were acceptable. Samples producing unreliable measures (i.e., intra-assay CVs >20%) of IL-6 (N=38; 4.8%) or CRP (N=22; 2.8%), even after being re-assayed in duplicate were excluded, leaving 753 and 769 measured data points for IL-6 and CRP, respectively.
Samples were centrifuged (1300 g) for 20 minutes and then immediately transferred into 2 mL tubes and stored at -80º C until the day of the assay.
IL-6 and CRP were assayed in duplicate using commercially available enzyme-linked immunosorbent assays (IL-6: Quantikine HS Human IL-6 ELISA, R&D Systems, Minneapolis, MN, USA; CRP: EIA-3954 High Sensitivity C-Reactive Protein ELISA DRG International Inc., USA). Intra-(IL-6: 5%, CRP: 4%) and inter-(IL-6: 14%, CRP: 13%) assay coefficients of variation were acceptable. Samples producing unreliable measures (i.e., intra-assay CVs >20%) of IL-6 (N=38; 4.8%) or CRP (N=22; 2.8%), even after being re-assayed in duplicate were excluded, leaving 753 and 769 measured data points for IL-6 and CRP, respectively.
9
Plasma IL-6 Quantification by ELISA
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Blood was collected into one 6 ml vacutainer containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant (Becton Dickinson Diagnostics, Oxford, United Kingdom). Samples
were immediately centrifuged at 1500g for 10 min at 4 °C and plasma was aliquoted and stored at -80 °C for later cytokine assessment of plasma interleukin-6 (IL-6). Plasma IL-6 was measured in duplicate using high-sensitivity enzyme-linked immunosorbent assay (ELISA) (Quantikine HS Human IL-6 ELISA, R&D Systems, UK) in accordance with the manufacturer's instructions. The limits of detection of this assay was .11 pg/mL, with an intra-assay coefficient of variation (CVs) of 4.2%. All samples from the same participants were assayed in the same run.
were immediately centrifuged at 1500g for 10 min at 4 °C and plasma was aliquoted and stored at -80 °C for later cytokine assessment of plasma interleukin-6 (IL-6). Plasma IL-6 was measured in duplicate using high-sensitivity enzyme-linked immunosorbent assay (ELISA) (Quantikine HS Human IL-6 ELISA, R&D Systems, UK) in accordance with the manufacturer's instructions. The limits of detection of this assay was .11 pg/mL, with an intra-assay coefficient of variation (CVs) of 4.2%. All samples from the same participants were assayed in the same run.
10
Plasma IL-6 Quantification by ELISA
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Blood (6 ml) was collected from an antecubital vein in the forearm into a vacutainer containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant (Becton Dickinson Diagnostics, Oxford, United Kingdom). Samples were immediately centrifuged at 1500g for 10 min at 4 °C and plasma was aliquoted and stored at -80 °C for later cytokine assessment of plasma interleukin-6 (IL-6) using high-sensitivity ELISA (Quantikine HS Human IL-6 ELISA, R&D Systems, UK) in accordance with the manufacturer's instructions. The limit of detection of this assay was 0.11 pg/mL, with an intra-assay coefficient of variation (CV) of 4.2%. All samples were well above the detection limit (the sample values ranged between 0.33 and 9.62 pg/mL). To minimize assay variation, all samples from the same participants were assayed in the same run..
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