To evaluate systemic cytokine/chemokine protein levels, we performed a custom 8-plex bead-based multiplexing assay using the Milliplex Human Cytokine/Chemokine Magnetic Bead Panel following the manufacturer’s recommendations (Millipore, Darmstadt, Germany) on plasma biospecimens from IR and IS individuals acquired during the same blood draws for PBMC. Antibodies for interferon gamma (IFN-γ), interleukin-10 (IL-10), IL-1β, IL-6, IL-8, TNF-α, MCP-1, and vascular endothelial growth factor (VEGF) were utilized in this custom 8-plex panel. Standards, controls 1 and 2, and samples were measured in duplicates. The human cytokine standards were customized to include a lower than recommended concentration (0.64 pg/mL) to increase the range of the standard curve for lower limits of detection. To further improve sensitivity of the assay for analytes, the plates were incubated overnight at 4 °C for 17 hours with constant agitation. Fluorescent signals were analyzed using the Luminex 200™ instrument (R&D Systems, Inc., Minneapolis, MN, USA). Bio-Plex Manager™ software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for data processing.
Luminex 200 instrument
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Luminex 200 instrument is a multiplex analysis system used for the detection and quantification of biomolecules, such as proteins and nucleic acids, in a sample. The instrument utilizes color-coded magnetic beads and fluorescent reporters to perform simultaneous measurements of multiple analytes in a single well. The Luminex 200 enables high-throughput and efficient analysis of samples, making it a valuable tool for researchers and clinicians.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex manager software, by Bio-Rad (2 mentions)
Luminex technology, by R&D Systems (2 mentions)
Milliplex human cytokine chemokine magnetic bead panel, by Merck Group (1 mentions)
Bio plex manager 6, by Bio-Rad (1 mentions)
Milliplex human cardiovascular disease panels, by Merck Group (1 mentions)
Elx405 washer, by Agilent Technologies (1 mentions)
Human magnetic luminex assay kit, by R&D Systems (1 mentions)
Luminex is software, by DiaSorin (1 mentions)
Luminex assay, by R&D Systems (1 mentions)
11 protocols using luminex 200 instrument
1
Quantifying Cytokine/Chemokine Profiles
2
Cytokine and Autoantibody Profiling
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The concentration of IL-6, IL-10 and TNFα were measured in all collected plasma samples in pg/ml using the Magnetic Luminex® Performance kit from R&D systems (Catalog number LUHM000) and analyzed on our Luminex 200 instrument. The cytokine panel was chosen in terms of viable indicators of the IL-6 signaling axis as well as thrombopoiesis. C-aAb against IL-1α, IL-6, IL-10, IFNα2, and GM-CSF were assessed using the aforementioned custom-made assay (19 (link)).
3
Profiling Cytokine Levels via Luminex
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Cytokine levels were measured by using the R&D Systems Human Premixed Multi-Analyte Kit Luminex Assay (Cat. no. LXSAH-6; R&D Systems, Minneapolis, MN, USA) and a Luminex 200 instrument (R&D Systems). Levels of IL-6, IL-8, IL-10, TNF-α, and VEGF were measured according to the manufacturer’s instructions. TGF-β levels were measured with the R&D Systems Magnetic Luminex Performance Assay and MAGPIX MILLIPLEX MAP instrument (MilliporeSigma, Danvers, MA, USA) according to the manufacturer’s instructions. Samples above the standard curve were considered to be maximum value, and samples under the curve sensitivity were annotated as 0. All data are displayed in pg/ml.
4
Multiplex Biomarker Analysis of HIV Patients
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Aliquots of plasma from 74 HIV-infected individuals on cART were used for multiplex biomarker analysis using Milliplex human cardiovascular disease panels (EMD Millipore, USA). Soluble biomarkers assessed included sE-selectin, sVCAM-1, sICAM-1, matrix metalloprotease 9 (MMP-9), myeloperoxidase (MPO), plasminogen activator inhibitor-1 (tPAI-1), C-reactive protein (CRP), serum amyloid A (SAA), serum amyloid P (SAP), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), IL-1β, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ). Standards and samples were measured in duplicate. Samples were acquired on a Luminex 200 instrument (R&D Systems, Inc.). Bio-Plex Manager 6.1 software (Bio-Rad Laboratories, Inc.) was used for data processing.
5
Serum Biomarker Profiling Protocol
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The serum levels of IgE and histamine were determined using the appropriate ELISA kits according to the manufacturer’s instructions. The serum levels of IL-13, IL-17A, IL-3, IL-31, IL-33, IL4, IL-5, and TSLP in the serum were measured using Luminex technology (R&D Systems, Abingdon, United Kingdom) on a Luminex 200 instrument.
6
Multiplex Cytokine Analysis in BAL
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BAL cytokines were measured using a custom multiplex immunoassay (eBioscience), according to the manufacturer’s protocol. Briefly, magnetic beads where incubated with undiluted BAL (performed with 1ml sterile and endotoxin-free 0.9% NaCl solution) samples in a 96-well plate for two hours, followed by washing and incubation with detection antibody (30 minutes). After washing, incubation with streptavidin-PE and another washing step, the plate was read on a Luminex 200 instrument (R&D Systems).
7
Quantitative Cytokine and Chemokine Profiling
8
Quantification of SARS-CoV-2 Antibodies
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The SeroFlash™ SARS-CoV-2 IgG/IgM ELISA Fast Kit (Epigentek Inc., Farmingdale, NY, USA) was used to quantify SARS-CoV-2 spike protein IgM and IgG antibodies in plasma samples. For further confirmation and characterization, the Human Coronavirus Ig Total 11-Plex ProcartaPlex™ Panel (Thermo Fisher) was used for screening of four anti-SARS-CoV-2 antibodies (spike trimer, S1 subunit, receptor binding domain (RBD), and nucleocapsid), and six anti-coronavirus strains (SARS-CoV-1, MERS, CoV-NL63, CoV-KHU1, CoV-229E, and CoV-OC43). Fluorescent signals were analyzed using a Luminex 200 instrument (R&D Systems, Inc., Minneapolis, MN, USA), and data processed using Bio-Plex Manager™ software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
9
Cytotoxicity of Chemotherapeutics in HCT116 Cells
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A total of 4 × 104 HCT116 or HCT116 p53−/− cells were plated per well of a 24-well plate and incubated 12–16 hours before treatment with 5-FU, CPT-11, oxaliplatin, cisplatin, or various combination treatments at the appropriate IC50 (combination treatments received each drug at their individual IC50). Cell supernatants were collected at 48 hours after treatment and stored at −20oC. Samples were prepared and run in triplicate on a Luminex 200 Instrument (R&D LX200-XPON-RUO). Sample preparation was conducted and instrument settings were selected based on the Human Magnetix Luminex Assay (R&D LXSAHM) protocol.
10
Colon Explant Cytokine Quantification
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Colons were flushed with PBS and cut open longitudinally. A small explant (approximately 15-20 mg) from the distal part of the colon was cultured for 6 h in 300 µl of IMDM complete (IMDMc) culture medium supplemented with 10% heat-inactivated FCS, 25 mM β-Mercapthoethanol and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin). Cytokine levels in the supernatants were quantified by Luminex technology (R&D Systems, Abingdon, UK) on a Luminex 200 instrument. Cytokine concentrations were calculated using the Luminex IS software (Luminex Corporation, Austin, TX), and were normalized to the respective colon weight.
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