To assess inflammation markers, serum samples were analyzed using mouse Magnetic Luminex Screening assays containing a premixed multi-analyte kit for murine TGF-β-1, TNF-α and IL-6 (R&D Systems, Minneapolis, MN, USA) on a Luminex 200 (Austin, TX, USA). The color generated was determined by measuring the optical density value at 450 nm with a spectrophotometric microtiter plate reader (Molecular Devices Corp., Sunnyvale, CA, USA).
Magnetic luminex screening assay
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Magnetic Luminex Screening Assay is a multiplex immunoassay platform that utilizes magnetic beads coated with capture antibodies to measure multiple analytes simultaneously in a single sample. The core function of this assay is to provide a convenient and high-throughput method for the quantitative analysis of a wide range of biological targets, including proteins, cytokines, and other biomolecules.
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Lab products found in correlation
Bio plex 200 system, by Bio-Rad (4 mentions)
Bio plex 200, by Bio-Rad (4 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Taqman advanced mirna cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Spectrophotometric microtiter plate reader, by Molecular Devices (1 mentions)
Luminex 200, by R&D Systems (1 mentions)
U 32012 centrifuge, by Boeco (1 mentions)
Duoset sandwich elisa, by R&D Systems (1 mentions)
Pherastar plate reader, by BMG Labtech (1 mentions)
Bio plex manager software version 5, by Bio-Rad (1 mentions)
Gibco penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Phenol red free mem, by Thermo Fisher Scientific (1 mentions)
Printex 90, by Evonik (1 mentions)
Qcl 1000 endpoint chromogenic lal assay, by Lonza (1 mentions)
Alamarblue reagent, by Thermo Fisher Scientific (1 mentions)
Proteome profiler cytokine array kit, by R&D Systems (1 mentions)
43 protocols using magnetic luminex screening assay
1
Multiplex Inflammation Marker Analysis
2
Biomarkers of Contrast-Induced Kidney Injury
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Whole blood and urine were collected at four time points: baseline (precontrast) and 24 hours, 48 hours, and 5 to 7 days after contrast administration. Blood and urine samples were centrifuged at 5000 rpm at 4°C for 10 and 2 minutes, respectively (U-32012 Centrifuge, Boeco, Germany), and the sera and urine were stored at −80°C until assayed. Concentrations of IL10, IL18, TNFα, NGAL, KIM1, and cystatin C were determined using Magnetic Luminex® Screening Assays (#LXSAHM-3, R&D Systems, Inc., Minneapolis, USA) in accordance with the manufacturer's instructions on the BioPlex™ 200 system (Bio-Rad, Texas, USA). The Bio-Plex manager software, version 5, was used for the determination of concentrations. Serum concentrations of β2M were determined by an enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc.). Serum creatinine was determined using the Jaffe method.
3
Determination of Plasma Cytokine Levels
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Processing of blood samples and determination of plasma concentrations is described elsewhere [20 (link)]. In short, peripheral blood samples collected in EDTA-anticoagulating tubes were spun down at 3000 rpm for 10 minutes immediately after collection after which plasma was stored at –80°C until batchwise analysis. An array of cytokines, adhesion molecules, and damage-associated molecular patterns was assessed, which are reported elsewhere. We here report only on plasma concentrations of the pro-inflammatory cytokines and receptors commonly reported to be associated with fatigue i.e., interleukin (IL)-6, IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor (TNF)-α, and soluble TNF receptor I (sTNF-RI)), all of which were assessed with Magnetic Luminex Screening Assays (R&D Systems, Minneapolis, MN). Cytokine concentrations were log-transformed to obtain normal distributions.
4
Multiplex Cytokine Profiling by ELISA and Luminex
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The concentration of TNF-α, IL-6, matrix metalloproteinase 9 (MMP-9), C-C motif chemokine ligand (CCL) 2, CCL3, CCL4, C-X-C motif chemokine ligand (CXCL) 1, CXCL2, CXCL5, and CXCL10 was determined using DuoSet sandwich ELISA and Magnetic Luminex Screening Assays (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s guidelines. Optical density absorbance was measured at 450 nm with correction at 570 nm using a Pherastar plate reader (BMG Labtech, Offenburg, Germany). Luminescence was measured with a Bio-Plex 200 System (Bio-Rad). The amount of each analyte was interpolated from the protein standard curve and multiplied with the appropriate dilution factor.
5
Cytokine and Immunoglobulin Profiles in Fasted Subjects
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Venous blood samples (5 x 2.5 ml whole blood) were drawn from the antecubital vein at rest in a sitting position in mornings after an overnight fast (~10 h). The subjects were instructed to avoid any strenuous exercise 48 h preceding the blood draw, and no exercise the day before. The concentrations of cytokines IL-1β, IL-1ra, IL-6, IL-10 and TNF-αin plasma were determined by Magnetic Luminex Screening Assays, according to the manufacturer’s instructions (R&D systems, Minneapolis, MN). However, IL-1β, IL-6, IL-10 and TNF-αwere all below detectible levels in our subjects and were not included in the results. Serum levels of IgM, IgG and IgA were measured by nephelometry. The inter- and intro-assay coefficients of variation of these measures are 0.81% and 1.51% for IgM, 0.88%and 6.1% for IgG, and 2.54% and 4.09% for IgA.
6
Serum Protein Biomarkers in Clinical Analysis
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Heat-inactivated (56°C for 30 min) serum from all patients and controls were analyzed for soluble protein biomarkers using proximity extension assay (PEA) technology (Olink AB, Uppsala, Sweden). The samples were analyzed using the inflammation (v.3022) panel, including a total of 92 analytes. Data are expressed as normalized protein expression (NPX) values. NPX is an arbitrary unit on a Log2 scale to normalize data to minimize both intra-assay and inter-assay variation.
Additionally, several soluble analytes were also measured in serum or plasma by use of custom made multiplex Magnetic Luminex Screening assays (R&D Systems), according to the manufacturer's instructions. Serum and plasma were diluted 1:2 prior to analysis in multiplex.
Analytes from Olink data and Magnetic Luminex Screening assays that had more than 33% and 25% of missing values, respectively, were excluded from analysis.
Left-censored data from the multiplex analysis were imputed using GSimp package 27 (link) in R (v. 3.6.0) 28 .
Additionally, several soluble analytes were also measured in serum or plasma by use of custom made multiplex Magnetic Luminex Screening assays (R&D Systems), according to the manufacturer's instructions. Serum and plasma were diluted 1:2 prior to analysis in multiplex.
Analytes from Olink data and Magnetic Luminex Screening assays that had more than 33% and 25% of missing values, respectively, were excluded from analysis.
Left-censored data from the multiplex analysis were imputed using GSimp package 27 (link) in R (v. 3.6.0) 28 .
7
Multiplex Cytokine Quantification
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Quantification was performed using Magnetic Luminex Screening Assays (R&D Systems, Inc, MN; for TNF-α (tumor necrosis factor-α), IL (interleukin)-1β, IL-6, ST2 [suppression of tumorigenicity 2], FS-7-associated surface antigen (FAS)-Ligand, and FAS) or Milliplex MAP panels (EMD Millipore Corporation, MA; for MMP -1, -2, and -9 & TIMP-1 and -4) according to manufacturers' guidelines. Assays were run using the Bio-Plex 200 system (Bio-Rad, Texas), and concentrations generated automatically using the Bio-Plex manager software, version 5.0 (Bio-Rad Laboratories Inc, Hercules).
8
Serum Biomarkers in Mouse Model
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After 70 days of feeding, mice were sacrificed by CO2 inhalation followed by exsanguination through cardiac puncture. The obtained serum was diluted 1:2 with saline solution, and the concentrations of cholesterol, HDL, LDL, lipase, CK and GOT were determined at the Institute for Clinical Chemistry of the MHH.
The levels of TNFα, leptin, FGF-21, MCP-1, IFNγ and IL-6 in diluted serum were measured using a Magnetic Luminex® Screening Assay (R&D Systems, Wiesbaden, Germany). The assay was performed according to the manufacturer’s instructions, and concentrations were determined by parallel standard curves for each parameter.
The levels of TNFα, leptin, FGF-21, MCP-1, IFNγ and IL-6 in diluted serum were measured using a Magnetic Luminex® Screening Assay (R&D Systems, Wiesbaden, Germany). The assay was performed according to the manufacturer’s instructions, and concentrations were determined by parallel standard curves for each parameter.
9
Cytotoxicity and Inflammatory Profiling of HTIW
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The HTIW under investigation are all in commercial production, but the manipulations were done specifically for this study by Morgan Advanced Materials (Bromborough, UK) and were provided to Heriot-Watt University in a coded form to allow in vitro experiments to be blinded. DQ12 quartz was donated by the Institute of Occupational Medicine (IOM) (Edinburgh, UK) (DQ12 was included in this study as a positive control for effects pertaining to crystalline silica); TiO2 was from Tioxide, UK; nanoparticle carbon black (NPCB) was obtained from Degussa (Printex 90). rhTNF-α was from Immunotools (Friesoythe, Germany); AlamarBlue reagent, Gibco™ Penicillin-Streptomycin, RPMI 1640, phenol red-free MEM were from Thermo Fisher (Paisley, UK); QCL-1000 Endpoint Chromogenic LAL Assay was from Lonza (Slough, UK); Proteome Profiler Cytokine Array Kit, Magnetic Luminex Screening Assay, and DuoSet ELISA kits were from R&D Systems (Abingdon, UK); 1-hydroxyl-2,2,6,6-tetramethyl-4-oxo-piperidine (Tempone-H) was from Enzo Life Sciences (Exeter, UK). All other substances used were obtained from Sigma-Aldrich (Poole, UK).
10
Cytokine Profiling of Colon Tissues
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Portions (~30 mg) of the RNA later-stored colon tissues were lysed in RIPA buffer containing proteases by sonification and 30 minutes of rotation at 4 °C. Aggregates were removed by centrifugation (20 min, 10,000× g, 4 °C) and protein concentration in the supernatants was measured using the Pierce BCA protein assay (Thermo Fisher Scientific). Quantification of cytokine concentrations in the supernatants was carried out using the Magnetic Luminex Screening Assay (R&D Systems, Minneapolis, MN, USA). The kit quantified the cytokines KC/CXCL1, MIP-2/CXCL2, MCP-1/CCL2, IL-12p70, IL-1β, IL-5, IL-10, IFNγ, TNF, IL-6, IL-17A, and IL-23p19.
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