Bio plex pro human cytokine 27 plex assay kit

Manufactured by Bio-Rad

Sourced in United States

The Bio-Plex Pro Human Cytokine 27-plex Assay kit is a multiplex immunoassay that allows for the simultaneous quantification of 27 different human cytokines and chemokines in a single well. The kit utilizes magnetic beads coated with specific antibodies to capture and detect the target analytes.

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Bio-Plex Pro Human Cytokine 27-plex Assay (100 citations) Bio-Plex assay (73 citations) Bio-Plex kit (41 citations) Bio-Plex Pro Human Cytokine 27-plex (23 citations) Bio-Plex ProTM Human Cytokine 27-plex Assay (13 citations) Bio-Plex Human Cytokine 27-plex assay (7 citations) Bio-plex ProTM Human Cytokine 27-plex assay kit (5 citations) Bio-Plex Pro Human 27-plex Assay kit (2 citations) Human Bio-Plex Pro (2 citations) Cytokine assay kits (2 citations)

23 protocols using bio plex pro human cytokine 27 plex assay kit

Cytokine Secretion in Macrophages upon LPS and IL-10 Treatment

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Macrophages were cultured on adherent plates for Bio-Plex assay (5 × 10⁴ cells/96-well) and analyzed for cytokine secretion 2, 6, and 24 h after LPS (100 ng/mL), or LPS (100 ng/mL) and IL-10 (100 ng/mL) treatment. Supernatants were collected for analyses and stored at −20 °C until use. Supernatants were analyzed for the presence of 27 different proteins with the Bio-Plex Pro™ human Cytokine 27-plex Assay Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.
For TNF-α secretion analyses, macrophages (5 × 10⁴ cells/96-well) were stimulated with LPS, or LPS and IL-10 for 6 h. Supernatants of stimulated macrophages were analyzed for TNF-α proteins with an enzyme-linked immunosorbent assay (ELISA) Kit (Invitrogen). The analysis was performed according to the manufacturer’s protocol with SpectraMax 340PC (Molecular Devices, San José, CA, USA).

Hoffmann D., Sens J., Brennig S., Brand D., Philipp F., Vollmer Barbosa P., Kuehle J., Steinemann D., Lenz D., Buchegger T., Morgan M., Falk C.S., Klein C., Lachmann N, & Schambach A. (2021). Genetic Correction of IL-10RB Deficiency Reconstitutes Anti-Inflammatory Regulation in iPSC-Derived Macrophages. Journal of Personalized Medicine, 11(3), 221.

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Secretome Analysis of Cytokines

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For secretome analysis, culture medium supernatants were collected during harvest. All the samples were stored at − 80 °C until analysis with Bio-Plex Pro Human Cytokine 27-plex Assay kit (Bio-Rad Laboratories) according to the manufacturer’s instructions using Bio-Plex 200 system (Bio-Rad). Data were analyzed with Bio-Plex Manager software version 4.1.1, and visualized utilizing R. In order to compare multiple assays, median fluorescence intensities (MFI) were first normalized (nFI) by subtracting the background measurement from each analyte. Principle coordinates were calculated in R in order to assess the degree of inter-assay technical variability in comparison with experimental, inter-cell-type variability. Due to the inter-assay variability, a reduced resolution, binned heatmap method was employed. The R package dr4pl v1.1.11³⁴ was used to create a combined standard curve from which the binning thresholds were established. Bins were drawn from the upper and lower asymptotes, as well as the calculated midpoint (EC50).

Gurvich O.L., Puttonen K.A., Bailey A., Kailaanmäki A., Skirdenko V., Sivonen M., Pietikäinen S., Parker N.R., Ylä-Herttuala S, & Kekarainen T. (2020). Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products. Scientific Reports, 10, 14049.

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Multiplex Serum Cytokine Analysis

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At the follow-up clinic visit, venous blood samples were collected in a fasting state from participants. The blood samples were then centrifuged, serum was aliquoted and frozen at −80°C until analysis. Circulating serum cytokine concentrations were determined by a multiplex immunoassay at the Australian Proteome Analysis Facility, using the Bio-Plex Pro Human Cytokine 27-plex Assay kit (Bio-Rad, Sydney, New South Wales, Australia). When the concentrations of any cytokine fell below the limit of detection in >25% of the study population, the cytokine was excluded from further analysis (IL-2, IL-15, and GM-CST). For the present study, a panel of 24 cytokines were analyzed (FGF basic, Eotaxin, G-CSF, IFN-γ, IL-1β, IL-1ra, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 [p70], IL-13, IL-17, IP-10 [CXCL-10], MCP-1 [MCAF], CCL3, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF).

Wu H., Mach J., Gnjidic D., Naganathan V., Blyth F.M., Waite L.M., Handelsman D.J., Le Couteur D.G, & Hilmer S.N. (2022). Comparing Effects of Polypharmacy on Inflammatory Profiles in Older Adults and Mice: Implications for Translational Aging Research. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 77(7), 1295-1303.

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Cytokine and MMP Response to Cigarette Smoke

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The proinflammatory properties of CS contribute to the initiation and progression of inflammatory respiratory diseases, including asthma and COPD (Strzelak et al. 2018 (link)). The inflammatory response triggered by CS has been correlated with MMP-mediated extracellular matrix remodeling in the lungs of smokers (Perlstein and Lee 2006 (link)). To assess these inflammatory responses to subacute exposures to CS, we quantified the secretion of select cytokines, chemokines, and MMPs into the basolateral medium at T12 and PT20. The Bio-plex Pro Human Cytokine 27-plex assay kit and a Human MMP 9-plex assay kit (Bio-Rad, Hercules, CA) were used for quantifying the secretion of select cytokines and MMPs into the basolateral medium. Briefly, basolateral media were incubated first with fluorescent magnetic beads by shaking vigorously at 850 ± 50 rpm for 1 h at room temperature, followed by incubation with the Detection Antibodies. The protein-antibody conjugates were visualized with streptavidin-PE. Fluorescence of the beads was measured and quantified using a MAGPIX system (Luminex, Austin, TX).

Xiong R., Wu Y., Wu Q., Muskhelishvili L., Davis K., Tripathi P., Chen Y., Chen T., Bryant M., Rosenfeldt H., Healy S.M, & Cao X. (2021). Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model. Archives of Toxicology, 95(5), 1739-1761.

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Multiplex Cytokine Quantification in Plasma and PBMC

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Twenty seven cytokines (including IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, FGF-β, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α and VEGF) in the plasma and culture supernatant samples were analyzed using Bio-Plex Pro Human Cytokine 27-plex Assay kit (Bio-Rad, Hercules, CA) according to the instruction manual. Briefly, the plasma or culture supernatant samples were mixed with the coupled beads in a 96-well filter plate and incubated on a shaker at room temperature for 30 min. After washing, the cytokine-coupled beads were mixed with the detection antibodies and incubated on a shaker at room temperature for 30 min. After washing again, the cytokine-coupled beads were mixed with streptavidin-PE and incubated on a shaker at room temperature for 10 min. After washing, the cytokine-coupled beads were resuspended in 125 µL of assay buffer. Next, data were acquired in the Bio-Plex Luminex 100 system and analyzed using Bio-Plex Manager Software version 5.0 (Bio-Rad, Hercules, CA) to determine 27 cytokines in plasma and cytokine production in supernatants of PBMC samples.

Zhou J., Nagarkatti P., Zhong Y., Ginsberg J.P., Singh N.P., Zhang J, & Nagarkatti M. (2014). Dysregulation in microRNA Expression Is Associated with Alterations in Immune Functions in Combat Veterans with Post-Traumatic Stress Disorder. PLoS ONE, 9(4), e94075.

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Biomarker Panel for Neurodegenerative Conditions

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A biomarker panel has been designed based on previous investigations by our group in older populations, including older adults with neurodegenerative conditions [10 (link),38 (link),39 (link),40 (link),41 (link),42 (link),43 (link)]. Markers of systemic inflammation will be assayed as described elsewhere [40 (link)]. Briefly, a set of 27 pro- or anti-inflammatory mediators (e.g., cytokines, chemokines, and growth factors) will be measured in serum samples using the Bio-Plex Pro™ Human Cytokine 27-plex Assay kit (#M500KCAF0Y, Bio-Rad, Hercules, CA, USA) (Table 2). Experiments will be run on a Bio-Plex^® System with Luminex xMap Technology (Bio-Rad) and data will be acquired on the Bio-Plex Manager Software 6.1 (Bio-Rad) with instrument default settings. Outliers will be automatically removed by optimization of standard curves across all analytes and results will be obtained as concentration (pg/mL).
Traditional and automated enzyme-linked immunosorbent assays will be run to quantify serum levels of amyloid beta (aa1-42) (#DAB142, R&D Systems, Minneapolis, MN, USA), neurofilament light polypeptide (#SPCKB-PS-002448-000190, R&D Systems), Tau (#NBP2-62749, Novus Biologicals, Littleton, CO, USA), and Tau [p Ser739] (#NBP2-66711, Novus Biologicals) proteins according to the manufacturer’s instructions.

Picca A., Ronconi D., Coelho-Junior H.J., Calvani R., Marini F., Biancolillo A., Gervasoni J., Primiano A., Pais C., Meloni E., Fusco D., Lo Monaco M.R., Bernabei R., Cipriani M.C., Marzetti E, & Liperoti R. (2020). The “develOpment of metabolic and functional markers of Dementia IN Older people” (ODINO) Study: Rationale, Design and Methods. Journal of Personalized Medicine, 10(2), 22.

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Quantifying Cytokine Levels in SFTS Serum

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We utilized the Bio-plex Pro Human cytokine 27-plex Assay kit (Bio-Rad, USA) to quantify the levels of cytokines in serum samples collected during the hospitalization of SFTS patients. This kit incorporates fluorescent microspheres that are conjugated with monoclonal antibodies that specifically target the cytokines of interest. To ensure accurate results, we strictly followed the instructions provided by the manufacturer, and all cytokines were tested using the same method. The following cytokines underwent testing: interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), Eotaxin, macrophage inflammatory protein 1β (MIP-1β), MIP-1α, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (basic FGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), inducible protein-10 (IP-10), granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-1β, IL-17, IL-15, IL-13, IL-12, IL-10, IL-9, IL-8, IL-7, IL-6, IL-5, IL-4, IL-2, IL-1 receptor antagonist (IL-1RA), regulated on activation and normally T-cell expressed (RANTES), and platelet-derived growth factor (PDGF-BB). Processed samples were subjected to testing using the Bio-Plex 3D system, which is a flow cytometry-based method.

Xu D.L., Zhang X.M., Tian X.Y., Wang X.J., Zhao L., Gao M.Y., Li L.F., Zhao J.Q., Cao W.C, & Ding S.J. (2024). Changes in Cytokine Levels in Patients with Severe Fever with Thrombocytopenia Syndrome Virus. Journal of Inflammation Research, 17, 211-222.

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Quantifying Cytokine and MMP Secretion

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The release of cytokines and MMPs into the basolateral medium was analyzed using a Bio-plex Pro Human cytokine 27-plex assay kit and a Human MMP 9-plex assay kit (Bio-Rad, Hercules, CA, USA), respectively, following the manufacturer’s instructions. Briefly, 50 µL basolateral media or analyte standards prepared in the PneumaCult™ Maintenance Medium were incubated with fluorescent magnetic beads for 1 h at 850 rpm ± 50 rpm in the dark. Unbound analytes were washed off by washing the plate three times with Wash Buffer. The analytes were then detected by incubating first with detection antibodies and then with Streptavidin-PE. The fluorescence of the beads was measured using a MAGPIX system (Luminex, Austin, TX, USA) and the intensity of the fluorescence was analyzed using the Bioplex Manager (Bio-Rad).

Wang Y., Wu Q., Ren B., Muskhelishvili L., Davis K., Wynne R., Rua D, & Cao X. (2022). Subacute Pulmonary Toxicity of Glutaraldehyde Aerosols in a Human In Vitro Airway Tissue Model. International Journal of Molecular Sciences, 23(20), 12118.

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Multiplex Cytokine Profiling in Fecal Samples

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The concentration of 27 cytokines, chemokines, and growth factors was determined using a Bio-Plex 200 system (Bio-Rad, Hercules, USA) and the Bio-Plex Pro Human Cytokine 27-plex Assay kit (Bio-Rad). Before analysis, faeces diluted tenfold (w/v) in PBS were centrifuged for 15 min at 20,000 g at 4 °C, and the supernatants were collected, diluted, and treated following the manufacturer’s protocol. Standards and samples were determined in duplicate. Data acquisition was performed with the Bio-Plex Manager 6.0 software and the standard curves fitted to a 5-parameter logistic regression.

Gutiérrez-Díaz I., Sanz-Martinez M., Castro A.M., Rodríguez-Belvís M.V., Carreira N., Jiménez S., Mangas C., Queralt M., Herrador M., Martín-Masot R., Ferrer P., Navas-López V.M., Espín B., Leis R., Díaz J.J, & Delgado S. (2023). Microbial and immune faecal determinants in infants hospitalized with COVID-19 reflect bifidobacterial dysbiosis and immature intestinal immunity. European Journal of Pediatrics, 182(10), 4633-4645.

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Modulation of Cytokine/Chemokine Response in Respiratory Cell Lines

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A549 and SAE cells were inoculated with HMPV, AdV, or RSV at a multiplicity of infection (MOI) of 1. After 2 h, cells were treated with dimethyl sulfoxide (DMSO, vehicle control), 20 μM CE3F4, or 20 μM MAY0132 in fresh media. At 15 h post-treatment, cytokines/chemokines from collected supernatant were quantified using a Bio-Plex Pro Human Cytokine 27-plex Assay kit from Bio-Rad according to the manufacturer’s instructions. Data were analyzed using the multiplex analysis software from Bio-Rad.

Choi E.J., Wu W., Cong X., Zhang K., Luo J., Ye S., Wang P., Suresh A., Ullah U.M., Zhou J, & Bao X. (2021). Broad Impact of Exchange Protein Directly Activated by cAMP 2 (EPAC2) on Respiratory Viral Infections. Viruses, 13(6), 1179.

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