The concentrations of C-reactive protein (CRP), a widely accepted inflammatory marker, were measured using competitive ELISA, previously validated for use in equine samples (Immunology Consultant Laboratories, Portland, OR, USA), from all treatment groups at time points 0, 8, and 24 h. A fluorescent bead-based multiplex assay (MILLIPLEX MAP Equine Cytokine/Chemokine Magnetic Beads Multiplex Assay, Millipore Sigma, Burlington, MA, USA) was used to quantify the concentrations of 23 analytes [eotaxin/CCL11, fibroblast growth factor 2 (FGF-2), fractalkine/CS3CL1, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), GRO, interferon (IFN), interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-17a, IL-18, IP-10, MCP-1, RANTES/CCL5, and tumor necrosis factor alpha (TNF-α)] in the SF from all time points.
Milliplex map equine cytokine chemokine magnetic beads multiplex assay
Manufactured by Merck Group
Sourced in United States
The Milliplex MAP Equine Cytokine/Chemokine Magnetic Beads Multiplex Assay is a laboratory equipment product that enables the simultaneous detection and quantification of multiple equine cytokines and chemokines in a single sample. The assay utilizes magnetic beads coated with specific antibodies to capture and measure the target analytes.
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4 protocols using milliplex map equine cytokine chemokine magnetic beads multiplex assay
1
Equine Inflammatory Marker and Cytokine Analysis
2
ELISA and Multiplex Assay for Cytokines
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An ELISA was used to measure the concentration of prostaglandin E2 (PGE-2 high sensitivity ELISA kit, Enzo Life Sciences, Inc. Farmingdale, NY 11735) and TGF-β (Human/Mouse/Rat/Porcine/Canine TGF-ß1 quantikine ELISA, R&D Systems, Inc. Minneapolis, MN 55413) in culture supernatants. Fluorescent bead-based multiplex assay (Milliplex MAP Equine Cytokine/Chemokine Magnetic Beads Multiplex Assay, Millipore Sigma, Burlington, MA, 01803) was used to quantify the concentrations of 23 analytes [Eotaxin/CCL11, FGF-2, Fractalkine/CS3CL1, G-CSF, GM-CSF, GRO, IFN, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12 (p70), IL-13, IL-17a, IL-18, IP-10, MCP-1, RANTES/CCL5 and TNFα] in cell culture supernatants.
3
Equine Cytokine/Chemokine Profiling
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Fluorescent bead-based multiplex assay (Milliplex MAP Equine Cytokine/Chemokine Magnetic Beads Multiplex Assay, Millipore Sigma, Burlington, MA, 01803) was used to quantitatively determine levels of 23 analytes (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-17a, IL-18, IP-10, MCP-1, RANTES/CCL5, TNF-α, Eotaxin/CCL11, FGF-2, fractalkine/CS3CL1, G-CSF, GM-CSF, GRO, IFN) in SF and plasma from all time points (d0, 1, 4, 7, 14). Quantification of SAA in SF and plasma was performed using SAA assay kit (StableLab, Epona Biotech Limited, Sligo, Ireland).
4
Equine MSCs Cytokine Secretion
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Equine MSCs from three donor horses were cultured in growth media with either 10% FBS or autologous or allogeneic equine serum for 72 h and then plated at 100,000 cells/well for 24 h on 24-well plates in media containing their respective serum sources. Supernatants were collected at 24 h and fluorescent bead–based multiplex assay (Milliplex MAP Equine Cytokine/Chemokine Magnetic Beads Multiplex Assay, Millipore Sigma, Burlington, MA) was used to quantify the concentrations of 23 analytes [eotaxin/CCL11, fibroblast growth factor 2 (FGF-2)]. Fractalkine/CS3CL1, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage CSF (GM-CSF), growth-regulated oncogene (GRO), interferon (IFN), interleukin 1α (IL-1α), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8/CXCL8, IL-10, IL-12, IL-13, IL-17a, IL-18, IP-10, monocyte chemoattractant protein 1 (MCP-1), RANTES/CCL5, and tumor necrosis factor (TNF-α) in conditioned media.
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