Milliplex mouse cytokine chemokine kit

Manufactured by Merck Group

Sourced in United States

The Milliplex Mouse Cytokine/Chemokine kit is a multiplex assay designed to detect and quantify multiple mouse cytokines and chemokines simultaneously in a single sample. The kit utilizes Luminex xMAP technology to enable the measurement of several analytes using a small sample volume.

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Lab products found in correlation

Bio plex 200 system, by Bio-Rad (6 mentions) Mouse cytokine chemokine array 32 plex, by Eve Technologies (1 mentions)

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12 protocols using milliplex mouse cytokine chemokine kit

Cytokine Profiling in Mouse Sera

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Sera from SMB19 Tg, J558 Tg, μMT, and C57BL/6J mice were collected at various time points before and after infection with A.f. Sera were diluted 3 fold and processed using the Milliplex Mouse Cytokine/Chemokine kit (Millipore). Prepared serum samples were run on the Luminex MAGPIX system and results analyzed on Milliplex Analyst software.

Wharton R.E., Stefanov E.K., King R.G, & Kearney J.F. (2015). Antibodies generated against streptococci protect in a mouse model of disseminated aspergillosis. Journal of immunology (Baltimore, Md. : 1950), 194(9), 4387-4396.

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Multiplex Cytokine Profiling in Mice

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We quantified 31 cytokine/chemokine/growth factor biomarkers simultaneously using a Milliplex Mouse Cytokine/Chemokine kit (Millipore, St. Charles, MO, USA) according to the manufacturers protocol. The multiplex was performed by Eve Technologies (Eve Technologies Corp, Calgary, AB, Canada) using the Bio-Plex 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The 31-plex consisted of eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF. The assay sensitivities of these markers range from 0.1–33.3 pg/mL. As IL-13 levels in the multiplex were mainly below detection, IL-13 protein levels were measured in the BAL fluid by an ELISA (eBioscience San Diego, CA, USA).

Bazett M., Biala A., Huff R.D., Bosiljcic M., Gunn H., Kalyan S, & Hirota J.A. (2016). A novel microbe-based treatment that attenuates the inflammatory profile in a mouse model of allergic airway disease. Scientific Reports, 6, 35338.

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Effects of Torilin on Cytokine Secretion

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RAW 264.7 cells were preincubated with torilin for 30 min before LPS stimulation for 24 h, and cytokine contents in the culture medium were measured by ELISA using anti-mouse TNF-α, IL-1β, IL-6, and GM-CSF antibodies and biotinylated secondary antibodies following the manufacturer's instruction (Millipore MILLIPLEX™ Mouse Cytokine/Chemokine kit (Millipore Corp., St. Charles, MO, USA)). In addition, PGE₂ contents in the culture medium were measured using prostaglandin E₂kit according to the manufacturer's instruction (Enzo life Sciences, Ann Arbor, MI, USA).

Endale M., Kim T.H., Kwak Y.S., Kim N.M., Kim S.H., Cho J.Y., Yun B.S, & Rhee M.H. (2017). Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation. Mediators of Inflammation, 2017, 7250968.

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Comprehensive Cytokine Profiling of BAL Fluid

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The Luminex multiplex assay was processed at Eve Technologies Corp using 50 μL BAL fluid per sample with the Mouse Cytokine Array/Chemokine Array 32‐Plex Discovery Assay® (MD31). The assay was analyzed on the Bio‐Plex™ 200 system (Bio‐Rad Laboratories, Inc.) using a Milliplex Mouse Cytokine/Chemokine kit (Millipore) according to the manufacturer's instructions. The 32‐plex consisted of 21 cytokines (IL‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐7, IL‐9, IL‐10, IL‐12 [p40 and p70], IL‐13, IL‐15, IL‐17, IFNγ, G‐CSF, GM‐CSF, M‐CSF, LIF and TNFα), ten chemokines (eotaxin/CCL11, IP‐10/CXCL10, KC/CXCL3, LIX/CXCL6, MCP1/CCL2, MIG/CXCL9, MIP‐1α/CCL3, MIP‐1β/CCL4, MIP‐2/CXCL2 and RANTES/CCL5), and vascular endothelial growth factor (VEGF). The sensitivities of these markers ranged from 0.1–33.3 pg/mL.

Ba M.A., Aiyuk A., Hernández K., Evasovic J.M., Wuebbles R.D., Burkin D.J, & Singer C.A. (2022). Transgenic overexpression of α7 integrin in smooth muscle attenuates allergen‐induced airway inflammation in a murine model of asthma. FASEB BioAdvances, 4(11), 724-740.

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Multiplex Cytokine Quantification in Sepsis

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Thirty-two different cytokines and chemokines were quantified simultaneously in plasma derived from mice in cohort 1 of the CLP model using the Mouse Cytokine/Chemokine Array 32-plex from Eve Technologies Corp (Calgary, Canada) using the Bio-PlexTM 200 system (Bio-Rad Laboratories, Hercules, CA, United States ), and a Milliplex Mouse Cytokine/Chemokine kit (Millipore, St. Charles, MO, United States ) according to the manufacturer’s protocols.

Cros C., Margier M., Cannelle H., Charmetant J., Hulo N., Laganier L., Grozio A, & Canault M. (2022). Nicotinamide Mononucleotide Administration Triggers Macrophages Reprogramming and Alleviates Inflammation During Sepsis Induced by Experimental Peritonitis. Frontiers in Molecular Biosciences, 9, 895028.

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Multiplex Analysis of Inflammatory Mediators

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We quantified 31 different mediators in the BALF and plasma by using a Discovery Assay® (Mouse Cytokine and Chemokine Array 31-Plex (MD31), Eve Technologies Corp, Calgary, AB, Canada). The multiplex assay was performed at Eve Technologies using the Bio-Plex™ 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a Milliplex Mouse Cytokine/Chemokine kit (Millipore, St. Charles, MO, USA) according to Eve Tech protocol (Eve Technologies 2021 ). The 31-plex consisted of eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF (leukemia inhibitory factor), LIX [lipopolysaccharide- induced CXC chemokines (CXCL5)], MCP-1, M-CSF (macrophage colony-stimulating factor), MIG (monokine induced by gamma interferon), MIP-1α, MIP-1β [macrophage inflammatory protein-1β (CCL4)], MIP-2, RANTES [regulated upon activation, normal T cell expressed and presumably secreted (CCL5)], TNF-α (tumor necrosis factor-alpha), and VEGF. The assay sensitivities of these markers range from 0.1 pg/mL to 33.3 pg/mL. Individual analytes’ values and other assay details are available on Eve Technologies’ website and in the Milliplex protocol.

Sordillo P.P., Allaire A., Bouchard A., Salvail D, & Labbe S.M. (2022). The complex lipid, SPPCT-800, reduces lung damage, improves pulmonary function and decreases pro-inflammatory cytokines in the murine LPS-induced acute respiratory distress syndrome (ARDS) model. Pharmaceutical Biology, 60(1), 1255-1263.

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Cytokine Profiling of Cabazitaxel-treated BMDMs

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BMDM cells were seeded in 24-well plates at a density of 5×10⁵ cells per well and incubated with DMSO (control) or cabazitaxel (10 µM). Cell culture medium without BMDM cells (negative control), DMSO-treated BMDM conditioned medium and cabazitaxel-treated BMDM conditioned medium were collected and centrifuged. The supernatants were collected and submitted to Eve Technologies for Mouse Cytokine Array/Chemokine Array 44-plex discovery arrays which were analyzed using Millipore MILLIPLEX mouse cytokine/chemokine kit.

Cao X., Li B., Chen J., Dang J., Chen S., Gunes E.G., Xu B., Tian L., Muend S., Raoof M., Querfeld C., Yu J., Rosen S.T., Wang Y, & Feng M. (2021). Effect of cabazitaxel on macrophages improves CD47-targeted immunotherapy for triple-negative breast cancer. Journal for Immunotherapy of Cancer, 9(3), e002022.

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Murine Airway Immune Profile Analysis

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Mice were anesthetized, and a 22-gauge cannula was inserted into the trachea of each mouse. Cells were collected by washing the airway lumen 3 times with 0.6 mL of saline. The supernatants were stored at −80°C until analysis. For differential cell count, cells were prepared using cytospin and stained with Diff-Quik (International Reagent Corp., Hyogo, Japan). Cytokine and chemokine concentrations in BALF were measured using a MILLIPLEX Mouse Cytokine/Chemokine kit (Millipore, Billerica, MA), which included eotaxin-1, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IP-10, KC, MCP-1, MIP-1α, MIP-2, RANTES, TNF-α, TGF-β1, TGF-β2, and TGF-β3.

Maki K., Nagai K., Suzuki M., Inomata T., Yoshida T, & Nishimura M. (2015). Temporal Changes in Glutaredoxin 1 and Protein S-Glutathionylation in Allergic Airway Inflammation. PLoS ONE, 10(4), e0122986.

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Cytokine Profiling of FLCN KO Cells

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RAW 264.7 cells were seeded in triplicates in 6-well plates at 1 × 10⁶ cells per well in DMEM medium supplemented with 10% FBS. After incubation for 24 h at 37 °C, 5% CO₂, cells were treated with LPS or GSK-621 or vehicle for 3, or 24 h, and the conditioned medium was harvested and centrifuged at 1,500 × g to remove cell debris. 32 cytokine/chemokine/growth factor biomarkers were simultaneously quantified by using a Discovery Assay® called the Mouse Cytokine Array/Chemokine Array 32-Plex (Eve Technologies Corp, Calgary, AB, Canada). The multiplex assay was performed by using the Bio-Plex™ 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and a Milliplex Mouse Cytokine/Chemokine kit (Millipore, St. Charles, MO, USA) according to the manufacturers protocol. The 32-plex consisted of Eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, VEGF. The change in the cytokine levels in FLCN KO medium was normalized against their respective wild-type medium.

El-Houjeiri L., Possik E., Vijayaraghavan T., Paquette M., Martina J.A., Kazan J.M., Ma E.H., Jones R., Blanchette P., Puertollano R, & Pause A. (2019). The transcription factors TFEB and TFE3 link the FLCN-AMPK signaling axis to innate immune response and pathogen resistance. Cell reports, 26(13), 3613-3628.e6.

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Multiplex Cytokine Analysis in BALF

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BALF total TGFβ1 was measured using a commercially available ELISA, according to the manufacturer’s protocol (R&D Systems, Cat# DY1679). We also quantified 32 different mediators in the BALF and cell culture supernatant by using a Discovery Assay® (Mouse Cytokine and Chemokine Array 32-Plex, Eve Technologies Corp, Calgary, AB, Canada). The multiplex assay was performed at Eve Technologies using the Bio-Plex™ 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a Milliplex Mouse Cytokine/Chemokine kit (Millipore, St. Charles, MO, USA) according to Evetech protocol. The 32-plex consisted of eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-13, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF. The assay sensitivities of these markers range from 0.1 pg/ml to 33.3 pg/ml. Individual analytes’ values and other assay details are available on Eve Technologies’ website and in the Milliplex protocol.

Ayaub E.A., Dubey A., Imani J., Botelho F., Kolb M.R., Richards C.D, & Ask K. (2017). Overexpression of OSM and IL-6 impacts the polarization of pro-fibrotic macrophages and the development of bleomycin-induced lung fibrosis. Scientific Reports, 7, 13281.

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