Levels of the cytokines IFNγ, tumour necrosis factor alpha (TNFα), IL-10, IL-17, IL-2, and IL-4 in culture medium were determined using a multiplex bead array assay. All the reagents were purchased from R&D Systems. Individual bead sets (Luminex) were coupled to cytokine-specific capture antibodies following the manufacturer’s instructions. Data were recorded on the Bio-Plex-200 platform and analysed using the Bioplex Manager® software (version 6.1; Bio-Rad) with a 5-parameter logistic regression algorithm.
Bio plex 200 platform
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Poland
The Bio-Plex 200 platform is a multiplex assay system capable of performing simultaneous detection and quantification of multiple analytes in a single sample. The system utilizes xMAP technology, which employs fluorescently dyed magnetic beads to capture and detect target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Luminex bead based immunoassays, by Merck Group (5 mentions)
Luminex xmap, by Bio-Rad (3 mentions)
Luminex xmap technology, by Bio-Rad (3 mentions)
Magnetic luminex assay, by R&D Systems (2 mentions)
Bio plex pro human cytokine, by Bio-Rad (2 mentions)
Bio plex manager 6, by Bio-Rad (2 mentions)
Manager 6, by Bio-Rad (2 mentions)
Bio plex manager software, by Bio-Rad (1 mentions)
Rat anti mouse ly6g 1a8, by BioXCell (1 mentions)
C57bl 6 mice, by Taconic Biosciences (1 mentions)
Procartaplex immunoassay, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Milliplex map, by Merck Group (1 mentions)
Durapore membrane, by Merck Group (1 mentions)
Masterscreen pneumo, by BD (1 mentions)
Interleukin 6 (il 6), by Merck Group (1 mentions)
Mcp 1, by Merck Group (1 mentions)
Tnf α, by Merck Group (1 mentions)
Il 1β, by Merck Group (1 mentions)
Hcytomag 60k, by Merck Group (1 mentions)
51 protocols using bio plex 200 platform
1
Multiplex Cytokine Profiling Assay
2
Multiplex Cytokine Profiling in Samples
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A Bio-Plex Multiplex Immunoassay System was used to measure 17 cytokine and chemokine (IL-6, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17A, MCP-1, MIP-1β, TNF-ɑ, G-CSF, GM-CSF, and IFN-γ) levels according to the manufacturer’s instructions. In brief, premixed beads were added to a 96-well plate from the kit. Plates were washed prior to adding 50 µl of standards and samples (4× dilution) to each well containing coated beads. The plate was placed on a shaker and incubated for 1 h at RT (850 RPM). Afterward, the plate was washed with wash buffer (3×) and detection antibodies were added to each well prior to incubating the plate again for 30 min on the shaker (850 RPM at RT). The plate was washed with wash buffer (3×) prior to adding streptavidin-PE (SA-PE) for 10 min. The plate was washed with wash buffer (3×) prior to measuring samples using the BioPlex 200 machine in the UAB Comprehensive Flow Cytometry Core. Data were analyzed using the BioPlex 200 platform.
3
Lung Cell and Cytokine Analysis
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Mice were sacri ced by CO 2 and lung tissue and blood samples were collected. Whole lungs were cyclically in ated and de ated with 1 mL of phosphate-buffered saline (Gibco-Thermo Fisher Scienti c, Waltham, MA, USA) three times. Cells were isolated by centrifugation at 300 × g for 10 min at 4°C and stained with Diff-Quik stain (Baso Diagnostics, Zhuhai, China). Differential cell counts were assessed from 400 cells counted on each slide. Plasma cytokine levels were assayed using Luminex xMap and a commercially available mouse cytokine 6-plex panel (Bio-Rad, Hercules, CA, USA) according to the manufacturer's guidelines and measured on a Bio-Plex 200 Platform.
4
Neutrophil Depletion in Pneumonia Mouse Model
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We used c57BL/6 mice (Taconic) aged 7 to 9 weeks for all mouse experiments, and pulmonary infection was performed as previously (14 (link), 67 (link)). At 48 and 4 h prior to the procedure, mice were injected intraperitoneally with 225 μg of rat anti-mouse Ly6G (1A8) or a control rat anti-mouse IgG2a (2A3) (BioXcell). Neutrophil depletion was confirmed previously using flow cytometry of lung homogenates (Ly6G+, Ly6C−, CD11b−). Inocula were prepared by resuspending MMC39 in a Tris-glycine buffer containing 5 mg/ml of an ovalbumin control mIgG1 (Crown Biosciences), 17H12 mIgG1, or 17H12 mIgG3 to a final concentration of 6 × 106 or 3 × 107 CFU/ml. After 1 h of opsonization, 50 μl of the inoculum was instilled into the surgically exposed trachea of a mouse under ketamine/xylazine using a bent 27-gauge needle. After 20 h, mice were euthanized, and lungs, liver, and spleen were collected and processed in NP-40 or PBS and diluted to enumerate CFU. Supernatants of lung homogenates used for cytokine analysis were stored at −80°C with 1× Pierce proteinase inhibitor until testing using Bio-Plex Pro mouse cytokine Th17 panel A with additional GM-CSF and IL-12p70 singleplex sets on a Bio-Plex 200 Platform (Bio-Rad). Cytokine levels were normalized against total protein measured by Bradford assay.
5
Quantifying Chemokine Levels in Cell Cultures
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For the purpose of the correlation analysis, the previously published [88 (link),89 (link)] data on circulating chemokines were obtained from the patients’ database. In the original research, concentrations of MCP-1, MIP-1α and MIP-1β were measured in sera in duplicates/triplicates using a flow cytometry-based method with a BioPlex 200 platform (Bio-Rad) and Bio-Plex Pro™ Human Cytokine, Chemokine, and Growth Factor Magnetic Bead–Based Assays, as instructed by the manufacturer.
For the purpose of this study, the concentrations of MCP-1, MCP-3, MIP-1α, and MIP-1β were measured in cell culture supernatants using the same methodology and a custom-made cytokine panel ProcartaPlex Immunoassay from Invitrogen (Thermo Fisher Scientific).
Standard curves were prepared using 5-PL logistic regression, and the collected data were analyzed using BioPlex Manager 6.0 software.
For the purpose of this study, the concentrations of MCP-1, MCP-3, MIP-1α, and MIP-1β were measured in cell culture supernatants using the same methodology and a custom-made cytokine panel ProcartaPlex Immunoassay from Invitrogen (Thermo Fisher Scientific).
Standard curves were prepared using 5-PL logistic regression, and the collected data were analyzed using BioPlex Manager 6.0 software.
6
Adiponectin, Leptin, and Resistin Levels
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Plasma levels of adiponectin, leptin, and resistin were measured in the fasting state using the human obesity multiplex Luminex assays (R&D Systems, Minneapolis, Minnesota, USA) and analyzed using a Bioplex 200 platform (Bio-Rad, Hercules, California, USA) from banked participant samples collected at baseline and final follow-up study visits. All samples were run in duplicates and analyzed according to the manufacturer’s instructions.
7
Cytokine Profiling of Proliferation Assays
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Supernatants collected from proliferation assays on day 2 and day 5 were assayed using a human cytokine/chemokine kit (Bio-Plex 14-plex; Bio-Rad Laboratories) on the Bio-Plex 200 platform according to the manufacturer’s instructions. Supernatants were selected on the basis of the largest proliferation responses per subject. Responses were considered positive if the value secreted was at least double the amount detected from unstimulated cells, and the background was subtracted before analysis.
8
Cytokine Quantification by Bioplex
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Samples were sterilised by filtration through a 0.22 μM Durapore membrane (MerkMillipore). Concentrations of cytokines (ThermoFisher, UK) were determined using a Bioplex 200 platform (Bio-Rad, UK) according to the manufacturer’s protocol and quantified per milligram of total protein measured by Bradford assay (Biorad).
9
Multiplex Cytokine Profiling of Immune Markers
10
Multiplex Cytokine Profiling in Serum
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Serum concentration of 27 cytokines, chemokines, and growth factors was quantified using the BioPlex 200 platform (Bio-Rad), incorporating Luminex xMAP® technology, allowing for simultaneous quantification of multiple analytes in real-time, and Bio-Plex Pro™ Human Cytokine, Chemokine, and Growth Factor Magnetic Bead–Based Assays. This flow cytometry-based method utilizes magnetic microspheres conjugated with monoclonal antibodies and fluorescent reading. All analyses were conducted in duplicates following manufacturer’s instructions. Standard curves were drawn using 5-PL logistic regression and the data were analyzed using BioPlex Manager 6.0 software.
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