Milliplex map human cytokine chemokine panel

We froze SOF, ACF, and VF and thawed them only once before applying the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay that allows the simultaneous quantification of the following human cytokines: fibroblast growth factor (FGF)-2, interferon (IFN)-γ, interleukin (IL)-10, IL-12p40, IL-1β, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF). We used values of “0” for samples under the detection sensitivity in the statistical analyses. We also measured electrolytes (Na, K, Cl, Ca, Fe, Mg, and Zn) using a LABOSPECT 008 (Hitachi High-Technologies Corporation, Tokyo, Japan) in SOF and VF and compared the average values.

Plasma samples were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay, which allows the simultaneous quantification of the following 29 human cytokines: IL-1α, IL-1β, IL-1 receptor antagonist (ra), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17A, EGF, Eotaxin, G-CSF, GM-CSF, IFN-α2, IFN-γ, CXCL10, MCP-1, MIP-1α, MIP-1β, TNF-α, TNF-β, and VEGF. Moreover, a custom made MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (Merck Millipore) was used to quantify the cytokines PDGF-AB/BB and RANTES (see Supplementary Table 1 available online at http://dx.doi.org/10.1155/2014/473765 for the list of the cytokines' abbreviations). Samples were processed in duplicate following the manufacturer's recommended protocols and read on a MAGPIX instrument equipped with the MILLIPLEX-Analyst Software using a five-parameter nonlinear regression formula to compute sample concentrations from the standard curves.

The cytokine analysis was performed using a MAGPIX instrument (Luminex Corporation, Texas, USA), xPONENT software (version 4.2.1324.0) and a MILLIPLEX MAP Human Cytokine/Chemokine Panel (EMD Millipore, Massachusetts, USA) detecting GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-α. The cytokine panel was designed to provide a measure of both Th1 and Th2 inflammatory response. All samples, standards and controls were run in duplicates and handled in accordance with the manufacturer’s protocol. Washing steps were performed twice. The system was set to analyse 100 µl of sample per well and collect a minimum of 100 beads per region.

Measurement of inflammatory cytokines (TNF-α, IL-1β, IL-2, and IL-6) in aqueous humor and opaque substance was performed employing a Luminex®200™ flow cytometer (Merck Millipore, Germany) multiple array assay system using Multiplex kits (Millipore) based on the Luminex xMAP technology designed to spontaneously measure multiple protein targets in a single sample at a very limited assay volume according to the manufacturer's instruction. The MILLIPLEX MAP Human Cytokine/Chemokine panel (EMD Millipore Corporation, Billerica, MA, USA) incubating 25 μl of the sample with antibodies at −4°C was used. Values were expressed as pg/ml, and the standard curve range from 3.2 to 10,000 pg/ml.

The biological samples were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay, which allows the simultaneous quantification of the following 29 human cytokines: EGF, IL-1β, IL-1 receptor antagonist (RA), IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, 1L-17A, EOTAXIN, G-CSF, GM-CSF, IFN-α2, IFN-γ, CXCL10, MCP-1, MIP-1α, MIP-1β, TNF-α, TNF-β, and VEGF. Samples were analysed in duplicate following the manufacturer's recommended protocols and the results were read on a MAGPIX instrument equipped with the MILLIPLEX-Analyst Software using a five-parameter nonlinear regression formula to compute sample concentrations from the standard curves, as previously described [18 (link)].

Serum samples were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay, which allows the simultaneous detection and quantification of the following 29 human cytokines/chemokines: EGF, Eotaxin, G-CSF, GM-CSF, IFN-α2, IFN-γ, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, 1L-17α, IL-1 receptor antagonist (ra), IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10 (CXCL10), MCP-1, MIP-1α, MIP-1β, TNF-α, TNF-β, and VEGF. Samples were processed following the manufacturer's recommended protocol and analyzed by using a MAGPIX instrument provided with the MILLIPLEX-Analyst Software that uses a five-parameter nonlinear regression formula to calculate cytokine/chemokine concentrations from the standard curves.

SOF and vitreous fluid were frozen and thawed only once before performing the MILLIPLEX MAP Human Cytokine/Chemokine Panel (Merck Millipore, Billerica, MA), a bead-based multiplex immunoassay which allows the simultaneous quantification of the following human cytokines: FGF-2, IFN-γ, IL-10, IL-12p40, IL-1β, IL-6, IL-8, MCP-1, TNFα and VEGF. Quantikine ELISA for human TGFβ1 (R&D Systems, Minneapolis, MN) was used to measure TGFβ1 following the manufacturer’s instruction. The values under detection sensitivity were defined as “0” in the statistical analyses.