Multiplex proteomic analysis was used to measure the levels of inflammatory cytokines, chemokines, and growth factors in the vitreous samples. The MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A–Immunology Multiplex Assay (HCYTA-60K-08; MilliporeSigma, Burlington, MA) was used for the MILLIPLEX enzyme-linked immunosorbent assay (ELISA). The concentrations of eight human mediators (IL-1β, IL-1α, IL-6, IL-8, IL-10, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor [GM-CSF]) were measured using a MAGPIX multiplex assay instrument (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Standard curves of known concentrations of recombinant human cytokines (R&D Systems, Minneapolis, MN) were used to convert fluorescence units to cytokine concentration (pg/mL). The protein concentration of each sample was determined using the bicinchoninic acid (BCA) method (G-Biosciences, St. Louis, MO).
Milliplex human cytokine chemokine growth factor panel a immunology multiplex assay hcyta 60k 08
Manufactured by Merck Group
The MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A–Immunology Multiplex Assay (HCYTA-60K-08) is a laboratory equipment product designed for the simultaneous quantification of multiple analytes in human biological samples. The assay uses bead-based technology to enable the detection and measurement of various cytokines, chemokines, and growth factors in a single sample.
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2 protocols using milliplex human cytokine chemokine growth factor panel a immunology multiplex assay hcyta 60k 08
1
Multiplex Analysis of Vitreous Mediators
2
Multiplex Cytokine Profiling in Plasma
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Multiplex enzyme-linked immunosorbent assays were performed on plasma of Cohort 1 for inflammatory mediators including cytokines and chemokines using a commercially available panel (MILLIPLEX® Human Cytokine/Chemokine/Growth Factor Panel A—Immunology Multiplex Assay HCYTA-60K-08, Millipore Sigma) by following the manufacturer’s instructions. The inflammatory mediators in the panel of the kit are: sCD40L, EGF, Eotaxin, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-1, MCP-3, M-CSF, MDC (CCL22), MIG, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNFα, TNFβ, and VEGF-A (see Additional file 2 for details).
Conventional ELISA was used to examine IL-6, IL-8, IL-10, TNF-α, and TGF-β1 in plasma of Cohort 2 patients at three time points (1 day, 7 days, 14 days) by using human ELISA LEGEND MAX (IL-6_430507, IL-8_431507, IL-10_430607, TNF-α_430207, and TGF-β1_436707; BioLegend). Signals were read at 450 nm in EnSpire™ Multimode Plate Reader (PerkinElmer, Inc.).
Conventional ELISA was used to examine IL-6, IL-8, IL-10, TNF-α, and TGF-β1 in plasma of Cohort 2 patients at three time points (1 day, 7 days, 14 days) by using human ELISA LEGEND MAX (IL-6_430507, IL-8_431507, IL-10_430607, TNF-α_430207, and TGF-β1_436707; BioLegend). Signals were read at 450 nm in EnSpire™ Multimode Plate Reader (PerkinElmer, Inc.).
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