Human cytokine magnetic 30 plex panel

We compared plasma levels of the following cytokines in Fil+ and Fil- subjects: epidermal growth factor (EGF), eotaxin, fibroblast growth factor (FGF)-basic, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), IFN-α, IFN-γ, IL-1ra, IL-1β, IL-2, IL-2r, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17, IP-10, monocyte chemoattractant protein (MCP)-1, monokine induced by IFN-γ (MIG, also known as CXCL9), macrophage inflammatory protein (MIP)-1α, MIP-1β, CCL5 (also known as RANTES, Regulated on Activation, Normal T Cell Expressed and Secreted), TNF-α, and vascular endothelial growth factor (VEGF). Samples were tested using the Human Cytokine Magnetic 30-Plex Panel (Invitrogen), and data were acquired with a Bio-Plex 200 System (Bio-Rad) using Luminex 100 xMAP technology (Luminex, Austin, USA).

To determine the concentration of a panel of inflammatory cytokines (EGF, Eotaxin, FGF basic, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17, IP-10, MCP-1, MIG, MIP-1α, MIP-1β, RANTES, TNF-α, and VEGF) in the patients’ sera, FlexMap3D luminex was applied. The analysis was performed using the Human Cytokine Magnetic 30-Plex Panel (Invitrogen, Poland) according to the manufacturer’s instructions. In total, 11 serum samples from sarcoidosis patients and five serum samples from healthy donors were tested. The results were analyzed with FlexMap3D xPONENT software.

As previously described (Pastor et al., 2017b (link)), CD4+ and CD8+ T cell counts were determined using CD3, CD8, CD4 and CD45 fluorochrome-labelled antibodies on fresh whole blood using Trucount tubes and FaCSCalibur flow cytometry (BD Biosciences, New Jersey). Cytokine levels in plasma were determined using enzyme-linked immunosorbent assay (Orgentec, Germany; Hycult Biotech, Pennsylvania; and Immunodiagnostik, Germany) and Luminex multianalyte profiling: Human Cytokine Magnetic 30-plex panel (Invitrogen, California), Bio-Plex Pro Human Th17 cytokine assay (Biorad, California), and Human Magnetic Luminex Screening Assay (R&D, Minnesota) (Pastor et al., 2017a (link)).

Plasma samples isolated from normal controls, OA patients, and SLE patients were analyzed within 6 months for a variety of cytokines, chemokines, cytokine receptors, and growth factors using the Human Cytokine Magnetic 30-Plex Panel (Novex®) (Invitrogen, USA) according to the manufacturer’s instructions, as previously described (11). Briefly, magnetic beads coupled with antibodies for 30 different analytes were added and washed twice with 1X wash buffer. The standard was prepared by mixing the 16plex and 14plex solutions provided by the manufacturer, and a serial dilution protocol was used to prepare a range of standard solutions (1:3 serial dilution). Both standards and serum samples (1:2 dilution) were prepared and added to the washed beads in the designated wells of the Mylar plates and incubated in an orbital shaker at 500 rpm for 2 h. Between incubations with different antibodies, the plate was washed twice with the wash buffer. The plate was then incubated with secondary antibodies for 1 h and with streptavidin-RPE-coupled detection antibodies for 30 min. The plate was washed three times, resuspended in wash buffer, and analyzed using a MAGPIX® instrument (Luminex Corporation, USA).

Cytokine levels were measured from plasma or nasal swab samples that included a baseline measurement per individual. Cohort plasma acute cytokine data were also used for a previous study^{14 (link)}. Nasal swabs were pre-diluted to 0.5 mg/ml for consistency with other protein analyses in this study. Cytokines were measured using the Human Cytokine Magnetic 30-Plex Panel (Invitrogen, Cat No. LHC6003M) and plates were read using a Luminex200 machine with xPONENT software (v4.3). Each sample was run in duplicate, and the average read was used for subsequent analyses. Sample exclusion from analyses included failure of detection for all cytokines and having no baseline value for comparison. Ingenuity pathway analysis (IPA) was used to identify pathways cytokines that were up or downregulated during the acute phase relative to baseline.