Human cytokine antibody array 5

Manufactured by RayBiotech

Sourced in United States

The Human Cytokine Antibody Array 5 is a multiplex assay designed to detect the simultaneous expression of multiple human cytokines in a single sample. The array consists of specific antibodies for 80 different cytokines, chemokines, and growth factors, allowing for comprehensive profiling of the cytokine expression profile in a biological sample.

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Lab products found in correlation

Image labtm software, by Bio-Rad (2 mentions) Image lab software, by Bio-Rad (1 mentions) Molecular imager chemidoc xrs, by Bio-Rad (1 mentions) Sphingosine 1 phosphate (s1p), by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions) Enhanced chemiluminescence ecl reagent, by GE Healthcare (1 mentions) Fluorescein isothiocyanate fitc conjugated secondary antibody, by Merck Group (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Sodium dodecyl sulfate (sds), by Merck Group (1 mentions) Protein assay, by Bio-Rad (1 mentions) Macrosep centrifugal devices, by Pall Corporation (1 mentions) Quantitative elisa, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

RayBio Human Cytokine Antibody Array 5 Cytokine arrays Antibody arrays

9 protocols using human cytokine antibody array 5

Cytokine Profiling of Fibroblast Secretome

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We used Human Cytokine Antibody Array 5 (RayBiotech, Norcross, GA, USA) to identify cytokines secreted by fibroblasts. Cytokine antibody membranes were incubated for 5 h with 1 mL of fibroblast conditioned media with or without adenocarcinoma cell co-culture. Membranes were incubated overnight with biotin-conjugated anti-cytokine antibodies, and then developed with horseradish peroxidase–streptavidin and chemiluminescence. The images were visualized using Molecular Imager ChemiDOC XRS+ (Bio-Rad, Hercules, CA), and quantified by Image Lab Software (Bio-Rad).
Recombinant human CXCL2, IL-8, and TNFRSF11B (osteoprotegerin, OPG) were purchased from BioLegend (San Diego, CA, USA). CXCL2, IL-8, and OPG were added into the medium. PD-L1 mRNA and protein expression were examined as described. We preliminarily examined the optimal concentration of CXCL2 in each cell line, and found that 100 ng/mL CXCL2 significantly suppressed the viability of PC-9. Therefore, we employed 10 ng/mL CXCL2 for PD-L1 induction in both PC-9 and H1975 cells. Otherwise, in A549, 100 ng/mL CXCL2 did not affect cell survival.

Inoue C., Miki Y., Saito R., Hata S., Abe J., Sato I., Okada Y, & Sasano H. (2019). PD-L1 Induction by Cancer-Associated Fibroblast-Derived Factors in Lung Adenocarcinoma Cells. Cancers, 11(9), 1257.

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Cytokine Expression Analysis by Antibody Array

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To investigate the expression of cytokines, cells in serum-free media (24 h) were collected and subjected to analysis by Human Cytokine Antibody Array 5 (RayBiotech, Norcross, GA, USA) according to the manufacturer’s instructions. The slide was analyzed using a GenPix 4000B scanner (Axon Instrument, Burlingame, CA, USA). The values were normalized and the background was reduced. The relative expression level of cytokines was determined by comparing signal intensities.

Chu H., Jia B., Qiu X., Pan J., Sun X., Wang Z, & Zhao J. (2017). Investigation of proliferation and migration of tongue squamous cell carcinoma promoted by three chemokines, MIP-3α, MIP-1β, and IP-10. OncoTargets and therapy, 10, 4193-4203.

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Cytokine Profiling of Stimulated Cells

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Cells were stimulated for 12 h with the indicated ligand, S1P or LPS from E. coli type 0111:B4 (Sigma, St. Louis, MO). Supernatants were analyzed with Human Cytokine Antibody Array 5 (RayBiotech, Norcross, GA), as described [19] (link). Cytokines analyzed include: ENA-78, G-CSF, GM-CSF, GRO, GRO alpha, I-309, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IFN-gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIG, MIP-1 beta, MIP-1 delta, RANTES, SCF, SDF-1, TARC, TGF beta 1, TNF alpha, TNF beta, EGF, IGF-1, Angiogenin, Oncostatin M, Thrombopoietin, VEGF-A, PDGF-BB, Leptin, BDNF, BLC, Ck beta 8-1, Eotaxin-1, Eotaxin-2, Eotaxin-3, FGF-4, FGF-6, FGF-7, FGF-9, Flt-3 ligand, Fractalkine, GCP-2, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF, Light, MCP-4, MIF, MIP-3 alpha, NAP-2, NT-3, NT-4, Osteopontin, Osteoprotegerin, PARC, PLGF, TGF beta 2, TGF beta 3, TIMP-1, and TIMP-2. Secretion of IL-6, IL-8, PGE₂, the soluble form of the intercellular adhesion molecule 1 (ICAM-1) and vascular endothelial growth factor (VEGF)-A was evaluated by immunoassay kits following the manufacturer's protocol (GE-Healthcare, Buckinghamshire, UK; RayBiotech, Norcross, GA). Absorbance was measured using a microplate reader Versamax (Molecular Devices, Sunnyvale, CA).

Fernández-Pisonero I., López J., Onecha E., Dueñas A.I., Maeso P., Crespo M.S., Román J.A, & García-Rodríguez C. (2014). Synergy between Sphingosine 1-Phosphate and Lipopolysaccharide Signaling Promotes an Inflammatory, Angiogenic and Osteogenic Response in Human Aortic Valve Interstitial Cells. PLoS ONE, 9(10), e109081.

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Investigating Cell Proliferation and Protein Expression

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Dulbecco’s modified Eagle’s medium (DMEM), Penicillin-streptomycin solution and Fetal bovine serum (FBS) were purchased from Gibco (Life technologies Korea, Seoul, Korea). 3-(4,5-dimethylhiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), Bicinchoninic acid (BCA) solution, Fluorescein isothiocyanate (FITC)-conjugated secondary antibody, Sodium dodecyl sulfate (SDS), Dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St, Louis, MO, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Santa-Curz Biotechnology (Santa Cruz, CA, USA). E-cadherin and MMP-1 antibodies were obtained through Cell Signaling Technology (Danvers, MA, USA). Enhanced chemiluminescence (ECL) reagent was obtained from GE Healthcare BIO-Sciences (Piscataway, NJ, USA). Anti-human CD4 FITC antibodies, Anti-human CD25 antibodies, Staining buffers were purchased from eBioscience (eBioscience, INC, San Diego, CA). Treg sol was provided by IMMUNISBIO. Co. Ltd. (Incheon, Korea) CD8+ T cell isolation kit was purchased from MACS (Miltenyi Biotec, Auburn, CA, USA). KBM502 medium was purchased from KOHJIN Bio (Salcado city, Saitama, Japan). Human cytokine antibody array 5 was purchased from RayBiotech (Raybiotech, Norcross, GA, USA).

Kim D., Lo E., Kim D, & Kang J. (2020). Regulatory T Cells Conditioned Media Stimulates Migration in HaCaT Keratinocytes: Involvement of Wound Healing. Clinical, Cosmetic and Investigational Dermatology, 13, 443-453.

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Cytokine Profiling of Conditioned Media

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Conditioned culture medium was frozen (-20°C), and cells were collected as described for Western blotting but with the cell pellets dissolved in RayBio^® Cell Lysis Buffer (RayBioech). Protein concentrations were determined using a protein assay (BioRad). Four membranes (Human Cytokine Antibody Array-5; RayBiotech) were incubated (30 min/room temperature) with blocking buffer (RayBiotech), and 1 ml conditioned culture medium or 160 μg cell lysate (diluted to 1 ml in blocking buffer) was added (incubation; 1 hr/RT followed by 12 hrs/4°C). After washing, membranes were incubated with biotin-conjugated antibody diluted in blocking buffer (2 hrs/room temperature and 12 hrs/4°C). Membranes were then incubated with horseradish peroxidase-conjugated streptavidin diluted in blocking buffer (2 hrs/room temperature), washed, developed with chemiluminiscence (RayBiotech), and visualized using a charged coupled device camera (Carestream). Densitometric analysis was performed using Image J software (NIH). Changes >50% relative to control were taken into consideration.

Dreyer-Andersen N., Almeida A.S., Jensen P., Kamand M., Okarmus J., Rosenberg T., Friis S.D., Martínez Serrano A., Blaabjerg M., Kristensen B.W., Skrydstrup T., Gramsbergen J.B., Vieira H.L, & Meyer M. (2018). Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells. PLoS ONE, 13(1), e0191207.

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Cytokine Profiling of hFOB Cells under AhR Ligand Treatment

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hFOB cells were cultured in a 100-mm culture dish. Culture medium was replaced by FBS and phenol red-free medium. 3-MC (10 μM) or β-NF (10 μM) was added, and after 24 h, conditioned medium (total 30 mL) was collected. AhR antagonist (10 μM CH-223191) and 3-MC were simultaneously added to the cell culture medium. Conditioned medium was concentrated to a 5 mL volume using Macrosep Centrifugal Devices (Pall Corporation, Port Washington, NY, USA). In this study, we employed the Human Cytokine Antibody Array 5 (RayBiotech, Inc., Norcross, GA, USA) in order to identify the cytokines released from hFOB cells with/without AhR ligand treatment. The membranes were incubated with biotin-conjugated anti-cytokines (provided with the kit) and developed with horseradish peroxidase-streptavidin and chemiluminescence. Protein dots were visualized with a Las-1000 cooled CCD-camera chemiluminescent image analyzer (Fuji Photo Film, Tokyo, Japan).

Miki Y., Hata S., Ono K., Suzuki T., Ito K., Kumamoto H, & Sasano H. (2017). Roles of Aryl Hydrocarbon Receptor in Aromatase-Dependent Cell Proliferation in Human Osteoblasts. International Journal of Molecular Sciences, 18(10), 2159.

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Cytokine Secretion in A549 and LK2 Cells

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As cortisol, hydrocortisone (HC) was purchased from MP Biomedicals (Solon, OH, USA). To assess the secretion levels of cytokines, we treated A549 and LK2 (1 × 10⁵ cells/ml) with ethanol as control or 100 nM HC for 24 h in the RPMI 1640 medium containing 10% FBS in six-well plates. After that, we removed the medium, washed the cells with PBS and incubated the cells in phenol red- and FBS-free RPMI 1640 medium for 24 h. The conditioned medium was used as samples. We used the Human Cytokine Antibody Array 5 (RayBiotech, Norcross, GA), which can detect 80 cytokines. The membranes were spotted with cytokine-specific antibodies and were analysed following the instructions from the manufacturer. The signal was detected using the Image Lab TM software (BIO‐RAD Laboratories, Inc., CA, USA).

Saito R., Miki Y., Abe T., Miyauchi E., Abe J., Nanamiya R., Inoue C., Sato I, & Sasano H. (2020). 11β hydroxysteroid dehydrogenase 1: a new marker for predicting response to immune-checkpoint blockade therapy in non-small-cell lung carcinoma. British Journal of Cancer, 123(1), 61-71.

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Quantifying Cytokine Levels via ELISA

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Levels of these substances were assessed using immunoassay array (Human Cytokine Antibody Array 5, RayBio, Norcross, GA, USA) and quantified by ImageJ software program (https://imagej.nih.gov/ij/). Levels of individual cytokines were determined by quantitative ELISA (Thermo Fisher).

Castellaro A.M., Rodriguez-Baili M.C., Di Tada C.E, & Gil G.A. (2019). Tumor-Associated Macrophages Induce Endocrine Therapy Resistance in ER+ Breast Cancer Cells. Cancers, 11(2), 189.

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Cytokine Secretion Profiling of miR-1 in PC9 Cells

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In this study, we treated PC9 (2.0 × 10⁵ cells/mL) with 50 nM miR‐1 for 24 h in the RPMI 1640 medium containing 10% FBS in order to further assess the potential effects of miR‐1 on secretion levels of cytokines. The medium was subsequently removed. The cells were then washed with phosphate‐buffered saline (PBS) and incubated in phenol red‐ and FBS‐free RPMI 1640 medium for 24 h. The conditioned medium was used in samples. We used the Human Cytokine Antibody Array 5 (RayBiotech, Peachtree Corners, GA, USA), which can detect 80 cytokines. The membranes were then spotted with cytokine‐specific antibodies and were analyzed. The signal was detected using the Image Lab TM software (BIO‐RAD Laboratories, Inc., Hercules, CA, USA).

Kawana S., Saito R., Miki Y., Kimura Y., Abe J., Sato I., Endo M., Sugawara S, & Sasano H. (2020). Suppression of tumor immune microenvironment via microRNA‐1 after epidermal growth factor receptor‐tyrosine kinase inhibitor resistance acquirement in lung adenocarcinoma. Cancer Medicine, 10(2), 718-727.

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