Plasma legumain concentrations were determined by enzyme immunoassay (EIA) with antibodies from R&D Systems (Duoset DY4769), Stillwater, MN. Serum levels of LDL-associated phospholipase A2 (Lp-PLA2) and autotaxin were determined by EIA from R&D systems (DPLG70 and DENP20, respectively). Plasma levels of tumor necrosis factor (TNF) was determined by a custom human cytokine kit, V-plex (cat # K151A0H-1), from Mesoscale Discovery, Rockville, MD. Plasma levels of platelet-factor (PF)4 (Cat# DY795), neutrophil-activating protein (NAP2/CXCL7) (Cat#DY393), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5) (Cat#DY278) and soluble CD40 ligand (CD40L) (Cat#DY617) were measured by EIAs obtained from R&D Systems. The intra-and inter-assay coefficients of variations were <10% for all EIAs. Routine biochemical analysis was performed in clinical routine laboratory at Oslo University Hospital.
Dplg70
Manufactured by R&D Systems
Sourced in United States
The DPLG70 is a laboratory equipment product manufactured by R&D Systems. It is a compact, lightweight device designed for general laboratory use. The core function of the DPLG70 is to provide a stable and reliable power source for a variety of laboratory applications.
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4 protocols using dplg70
1
Plasma Biomarker Profiling in Research
2
Biomarkers in Ischemic Stroke Patients
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Clinical information and cerebrovascular risk factors were collected by an interviewer, including personal and family history of hypertension, hyperlipidemia, diabetes and coronary heart disease, smoking and drinking habits, current drug intake, among others. Each patient's initial National Institute of Health Stroke Scale (NIHSS) was assessed at admission. All biochemical measurements, including complete blood count, serum lipid levels, liver and kidney functions, fasting plasma glucose, homocysteine (HCY), and erythrocyte sedimentation rate (ESR) were performed at the Central Laboratory of Xuanwu Hospital (Beijing, China) using standard protocols.
Blood samples were collected within 24 h of admission. Serum was separated by centrifugation for 10 min at 2,000 × g and stored in freezers at −80°C. To reduce inter-assay errors, Lp-PLA2 and high-sensitivity C-reactive protein (hs-CRP) levels were measured simultaneously for all participants using an enzyme-linked immunoassay according to the manufacturer's instructions. In brief, the serum was added to a microtiter plate that was pre-immobilized with a monoclonal antibody against Lp-PLA2 or hs-CRP (DPLG70, QK1707, R&D System, USA) and then incubated with anti-Lp-PLA2 or anti-hs-CRP antibody labeled with horseradish peroxidase. Optical density was determined with a microplate reader at 450 nm.
Blood samples were collected within 24 h of admission. Serum was separated by centrifugation for 10 min at 2,000 × g and stored in freezers at −80°C. To reduce inter-assay errors, Lp-PLA2 and high-sensitivity C-reactive protein (hs-CRP) levels were measured simultaneously for all participants using an enzyme-linked immunoassay according to the manufacturer's instructions. In brief, the serum was added to a microtiter plate that was pre-immobilized with a monoclonal antibody against Lp-PLA2 or hs-CRP (DPLG70, QK1707, R&D System, USA) and then incubated with anti-Lp-PLA2 or anti-hs-CRP antibody labeled with horseradish peroxidase. Optical density was determined with a microplate reader at 450 nm.
3
Biomarker Quantification in Stroke Patients
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The median time of sampling was 55 h (interquartile range: 27–96 h) after index event onset. Specimens were extracted, aliquoted and transported through cold chain to the core laboratory in Beijing Tiantan Hospital. All specimens were stored at − 80 °C until assays were performed centrally and blindly. The concentrations of IL-6, IL-1Ra, Lp-PLA2 and YKL-40 were determined by using enzyme-linked immunosorbent assay kits (catalogue number: PHS600C for IL-6, PDRA00B for IL-1Ra, DPLG70 for Lp-PLA2 and DC3L10 for YKL-40, R&D Systems, Inc, Minneapolis, MN, USA). Lp-PLA2-A was measured with an automatic enzyme assay system on a Hitachi 7600 analyzer (PLAC test for Lp-PLA2-A, Diazyme Laboratories, Inc., Poway, CA), and hsCRP and LDL-C were detected on Roche Cobas C501 and C702 analyzers, respectively.
4
Modulation of Monocyte Cytokines by PAF-AH
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Recombinant PAF-AH was dissolved in 0.5 ml of PBS and further diluted in DMEM media (2.5-10 ng/ml). Freshly isolated monocytes were pre-treated with recombinant PAF-AH for 20 min prior to the addition of MSU crystals and then incubated at 37˚C for 24 h after which supernatants were collected for cytokine analysis.
Enzyme linked immunosorbent assays (ELISAs). IL-1β and IL-6 concentrations in cell culture supernatants were determined by sandwich ELISA using matched antibodies (DuoSet: R&D Systems, Abingdon, UK). PAF-AH levels were determined using a solid phase quantikine ELISA (DPLG70 from R&D Systems) with sensitivity (0.8-50 ng/ml) in conditioned cell culture supernatants. All samples were measured in duplicates using manufacturer's instructions without any deviations. Results were expressed as the mean ± SEM cytokine concentration (ng/ml) from at least 4 experiments.
Statistical analysis. All samples were measured in duplicate from at least three experiments, with results expressed as the mean ± standard deviation. Statistical analysis was carried out by applying one-way analysis of variance with Dunnett's post hoc test or Student's t-test where appropriate. P<0.05 was considered to indicate a statistically significant difference. All data were analysed using Excel (2016 version; Microsoft Corporation, Redmond, WA, USA).
Enzyme linked immunosorbent assays (ELISAs). IL-1β and IL-6 concentrations in cell culture supernatants were determined by sandwich ELISA using matched antibodies (DuoSet: R&D Systems, Abingdon, UK). PAF-AH levels were determined using a solid phase quantikine ELISA (DPLG70 from R&D Systems) with sensitivity (0.8-50 ng/ml) in conditioned cell culture supernatants. All samples were measured in duplicates using manufacturer's instructions without any deviations. Results were expressed as the mean ± SEM cytokine concentration (ng/ml) from at least 4 experiments.
Statistical analysis. All samples were measured in duplicate from at least three experiments, with results expressed as the mean ± standard deviation. Statistical analysis was carried out by applying one-way analysis of variance with Dunnett's post hoc test or Student's t-test where appropriate. P<0.05 was considered to indicate a statistically significant difference. All data were analysed using Excel (2016 version; Microsoft Corporation, Redmond, WA, USA).
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