Plasma measurements were performed from blood samples collected at age 18 months following an overnight 12-hour fast. Insulin, C-peptide, interleukin-6 (IL-6), resistin, leptin, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor α (TNF-α) were measured using a mouse metabolic magnetic bead panel (RRID: AB_2801416; catalog No. MMHMAG-44K; MilliporeSigma) and a Luminex 200 System (catalog No. 40-012; MilliporeSigma) as previously described (38) (link). Adiponectin was measured using a mouse high-molecular-weight and total adiponectin enzyme-linked immunosorbent assay kit (RRID: AB_2801466; catalog No. 47-ADPMS-E01; ALPCO) according to the manufacturer's instructions, with 7 to 11 mice per group.
Luminex 200 system
Manufactured by Merck Group
Sourced in United States, Germany, Japan, Morocco
The Luminex 200 system is a flow cytometry-based platform designed for multiplexed analysis of biological samples. The system utilizes color-coded magnetic microspheres to simultaneously detect and quantify multiple analytes in a single sample. The core function of the Luminex 200 system is to perform high-throughput, quantitative measurements of proteins, nucleic acids, and other biomolecules.
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Lab products found in correlation
17 plex mouse procartaplex panel, by Thermo Fisher Scientific (3 mentions)
Milliplex map mouse cytokine chemokine magnetic bead panel, by Merck Group (3 mentions)
Xponent software, by Merck Group (2 mentions)
Mbnmag 41k, by Merck Group (2 mentions)
Fgf21, by Merck Group (2 mentions)
Milliplex xmap mouse high sensitivity tcell magnetic bead panel kit mhstcmag 70k, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
47 adpms e01, by ALPCO (1 mentions)
Mmhmag 44k, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Ifn α, by Thermo Fisher Scientific (1 mentions)
Cxcl10, by Thermo Fisher Scientific (1 mentions)
Luminex xmap technology, by Merck Group (1 mentions)
Quantikine human il 6 immunoassay, by R&D Systems (1 mentions)
Milliplex mouse cytokine chemokine magnetic bead panel, by Merck Group (1 mentions)
Bio plex pro human il 6 assay, by Bio-Rad (1 mentions)
Bio plex manager software version 6, by Bio-Rad (1 mentions)
Bio plex pro 2 wash station, by Bio-Rad (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
71 protocols using luminex 200 system
1
Metabolic Biomarker Measurements in Mice
2
Multiplex Cytokine Profiling
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Cytokine levels were analyzed by cytometric bead array. For human samples IFNα, IFNγ, CXCL10, and IL6 were measured (ThermoFisher Scientific). For macaque samples, IFNγ, IL8, IL10, IL15, and IL17 were measured using a custom kit from Millipore Sigma (Burlington, MA). The manufacturer’s recommended protocol was followed, and plates were read on Luminex 200 system (MilliporeSigma).
3
MHC Binding and Immunogenicity Predictions
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H-2Kd-restricted MHC class I binding and immunogenicity predictions were performed using syfpeithi. de, NeTMHC3.4, and Immune Epitope Database (IEDB) Analysis Resource (Kim et al., 2012 (link); Lundegaard et al., 2008 (link); Nielsen et al., 2003 (link); Peters and Sette, 2005 ; Rammensee et al., 1999 ; Sidney et al., 2008 ). The selected peptides (FFGAIAGFL (P1) and YYSTAASSL (P2)), where P1 is absent from rAd-HA2ΔN30F and P2 is absent from rAd-HA2ΔTMF, were synthesized with over 95% purity (New England Peptides). Four million spleen cells were then stimulated with 10 ug/ml of each of the selected peptides. Following a 48-hour incubation, secreted cytokines levels in the supernatant were measured using a ProcartaPlex Multiplex Immunoassay kit (Life Technologies). The plates were read on a Luminex 200 system (MilliporeSigma). Data analysis was performed using MILLIPLEX Analyst version 5.1 software for determining pg/ml of each cytokine.
4
Multiplexed Cytokine Profiling of Patient Plasma
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Cryopreserved patient plasma was sent to Eve Technologies Corp. for quantification of human cytokines, chemokines, and growth factors. Cytokine Luminex xMAP technology was used for multiplexed quantification of 71 human cytokines, chemokines, and growth factors: 6CKine, BCA-1, CTACK, EGF, ENA-78, eotaxin, eotaxin-2, eotaxin-3, FGF-2, Flt3L, fractalkine, G-CSF, GM-CSF, GROα, I-309, IFNα2, IFNγ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-16, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-28, IL-33, IP-10, LIF, MCP-1, MCP-2, MCP-3, MCP-4, M-CSF, MDC, MIG, MIP-1α, MIP-1β, MIP-1δ, PDGF-AA, PDGF-AB/BB, RANTES, sCD40L, SCF, SDF-1α+β, TARC, TGFα, TNFα, TNFβ, TPO, TRAIL, TSLP, and VEGF-A.
The multiplexing analysis was performed using the Luminex 200 system with assay kits sourced by Millipore MILLIPEX (MilliporeSigma) according to the manufacturer’s protocol. Observed concentrations were calculated with the standard curve based on the fluorescence intensity of the bead population for a specific analyte. Observed concentrations for each analyte were averaged across every cohort and scaled across cohorts with values ranging from −2 to 2 across all analytes. Heatmap visualizations were generated using the Python data visualization library pheatmap (Kolde, 2012 ).
The multiplexing analysis was performed using the Luminex 200 system with assay kits sourced by Millipore MILLIPEX (MilliporeSigma) according to the manufacturer’s protocol. Observed concentrations were calculated with the standard curve based on the fluorescence intensity of the bead population for a specific analyte. Observed concentrations for each analyte were averaged across every cohort and scaled across cohorts with values ranging from −2 to 2 across all analytes. Heatmap visualizations were generated using the Python data visualization library pheatmap (Kolde, 2012 ).
5
Multiplex Screening of GC-MSC Secretome
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Multiplex screening assays for GC-MSCs supernatants were performed using Human Cytokine/Chemokine Panel 1 according to the protocol provided by the manufacturer (Millipore, Billerica, MA, USA). Data were analyzed using the Luminex 200 system (Millipore). IL-6 levels in GC-MSCs supernatants were determined using ELISA kit according to the manufacturer's instructions (Dakewe, Beijing, China).
6
Quantification of Murine IL-6 Levels
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IL-6 concentrations in mouse serum were quantified using the MILLIPLEX mouse cytokine/chemokine magnetic bead panel (EMD Millipore) according to the manufacturer's instructions. Samples were incubated with antibody-coupled beads overnight. Washing procedures were performed using the ELx405 wash station (BioTek) and fluorescence intensity was detected by a Luminex 200 System in combination with xPONENT Software version 3.1 (Millipore). IL-6 concentrations in hepatocyte supernatants were measured using the Bio-Plex Pro human IL-6 assay in combination with the Bio-Plex Pro reagent kit (both Bio-Rad) according to the manufacturer's instructions. A dilution series of the recombinant human IL-6 used for stimulation was used as standard curve. Washing was performed using the Bio-Plex Pro II wash station (Bio-Rad) and fluorescence intensity was acquired using the Bio-Plex 200 system and Bio-Plex Manager software version 6.1 (both Bio-Rad). Alternatively, IL-6 concentrations in hepatocyte supernatants were determined using the Quantikine Human IL-6 Immunoassay (R&D Systems). Stabilization of ligand in medium samples was achieved by supplementing 450 μL conditioned medium with 50 μL of 40 mM HCl and 10 mg/mL BSA.
7
Cytokine Profiling Using Luminex
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The levels of plasma cytokines (interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1α, MIP-1β, and MIP-2) were measured using Luminex kits (MAGPIX; Millipore, Darmstadt, Germany) and analyzed using the Luminex 200 system with xPONENT software (Millipore) according to the manufacturer’s instructions.
8
Canine Hydrodynamic Injection Protocol
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Blood samples were collected from each dog before (time = 0), and 2, 4, 24, 48, and 96 h after either hydrodynamic injection or slow injection (1,000 ml) in 1 or 2 h through the peripheral vein as controls. The serum biochemical analysis and hemogram were performed by the Cleveland Office of Marshfield Labs and BML Inc. (Shibuya-ku, Tokyo, Japan). The cytokines were analyzed by the GeneticLab. Co. Ltd. (Sapporo, Hokkaido, Japan) using the Luminex 200 System with the MILLIPLEXMAP Canine Cytokine/Chemokine Kit (MILLIPORE, CCYTO-90K) [18] (link), [19] (link). Tissue samples for hematoxylin and eosin (H&E) staining were collected before, immediately following, and 3 h after the hydrodynamic injection to rats, and before, immediate following, and 24 h after the injection to dogs. Three different liver sections from each of the 3 rats and 2 dogs were stained, images were captured, and a quantitative analysis of the sinusoidal area was performed using ImageJ software (version 1.6.0_20, National Institutes of Health, USA) as previously reported [20] (link).
9
Quantification of Cytokines and TNF-α in Cell Supernatants
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Cytokines were quantified in AM supernatants using a Milliplex MAP Human Cytokine Kit (Millipore, #HCYTOMAG-60K) as detailed in Hoppstädter et al. (23 (link)). AMs were kept at a density of 1 × 105 cells per well in 96 well plates in a total volume of 100 μL medium in the presence or absence of Pam3CSK4 (100 ng/mL) or Poly(I:C) (10 μg/mL) for 6 h. The supernatants were collected and stored at −80°C until they were used in the multiplex cytokine assay. The immunoassay procedure was performed using a serial dilution of the 10,000 pg/mL human cytokine standard according to the manufacturer's instructions, and the plate was read at the Luminex 200 System (Millipore).
Murine TNF-α was quantified by bioassay as previously described (11 (link)). L929 cells were seeded at a density of 3 × 104 cells per well into a 96-well plate. After 24 h, the medium was replaced by 100 μL of actinomycin D solution (1 μg/mL in standard medium) and cells were incubated for 1 h at 37°C. Subsequently, BMM supernatants (100 μL per well) were added. Dilution series of recombinant murine TNF-α (100–2,500 pg/mL) were run alongside the samples to generate a standard curve. The plates were incubated for an additional 24 h at 37°C. The MTT assay (see Determination of Cell Viability) was used to assess cell viability.
Murine TNF-α was quantified by bioassay as previously described (11 (link)). L929 cells were seeded at a density of 3 × 104 cells per well into a 96-well plate. After 24 h, the medium was replaced by 100 μL of actinomycin D solution (1 μg/mL in standard medium) and cells were incubated for 1 h at 37°C. Subsequently, BMM supernatants (100 μL per well) were added. Dilution series of recombinant murine TNF-α (100–2,500 pg/mL) were run alongside the samples to generate a standard curve. The plates were incubated for an additional 24 h at 37°C. The MTT assay (see Determination of Cell Viability) was used to assess cell viability.
10
Multiplex Cytokine Analysis in Cell Culture
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Using a multiplex system of analysis, 25 μL of the culture supernatants were employed using the MILLIPLEX MAP mouse cytokine/chemokine panel (MCYTOMAG-70K; Millipore Corporation) and Luminex 200™ System (Millipore Corporation) according to the manufacturer’s instructions. Then, four analytes were studied: IL-2, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α). The concentration of analytes was determined by the xPONENT® software (Millipore Corporation), and the results are reported as the mean ± S.E.M. of cytokines (pg/mL).
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