Bio plex 200 system instrument

Twenty-seven plasma cytokines, chemokines, and growth factors were analyzed using the Bio-Plex Pro Human Cytokine 27-plex panel (Bio-Rad Laboratories Inc, Hercules, CA, USA) following the manufacturer instructions. Prior to quantification, plasma samples were diluted 1 : 4 in standard diluent (provided by the manufacturer). By using such panel, serum samples were screened for interleukin (IL)-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, eotaxin, basic fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon (IFN)-γ, IFN-γ induced protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1 α/β (MIP-1α, MIP-1β), platelet-derived growth factor (PDGF), RANTES (regulated on activation normal T-cell expressed and secreted), tumor necrosis factor α (TNF-α), and vascular endothelial growth factor (VEGF) concentrations. Fluorescent signals were recorded using a Bio-Plex 200 System instrument and analyzed using the Bio-Plex Manager Software (Bio-Rad Laboratories Inc, Hercules, CA, USA). The software fitted samples' median fluorescence intensity (MFI) values versus standards' MFI and converted it to concentration (pg/mL) by applying a five-parameter logistic regression (as suggested by the manufacturer).

TNF-α, IL-17, IL-1β, IL-6, and IFN-γ were quantified in serum using a convenient bioplex kit assay, according to the manufacturer’s instructions. The concentrations of cytokines were measured using Bio-Plex™ 200 System instrument (Bio-Rad Laboratories, Hercules, CA, USA) and Bio-Plex manager 4.1 Software.

IL-6 and TNF-α were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Results were measured at an optical density of 450 nm (reference wavelength 630 nm), and concentrations in pg/mL were derived from the standard curve. IL-1β, IL-10, Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES) and MCP-1 were measured using a multiplex bead array kit according to the manufacturer’s instructions (Bio-Rad) with results analyzed on a Bio-Plex 200 system instrument (Bio-Rad) fitted with the Bio-Plex Manager v6 software (Bio-Rad) and results reported in pg/mL.

Concentrations of Igs (IgA, IgG, and IgM), transforming growth factor-beta 2 (TGF-β₂) and hormones (adiponectin, ghrelin, and leptin) were determined in duplicate using a Bioplex 200 system instrument (Bio-Rad, Hercules, CA, USA) and the Bio-Plex Pro Human Isotyping Assay, Bio-Plex Pro Human TGF-β Assay and Bio-Plex Pro Human Diabetes Assays kits (Bio-Rad). The concentration of EGF was measured using the RayBio® Human EGF ELISA kit (RayBiotech, Norcross, GA). Every assay was performed according to manufacturer's instructions. Standard curves were performed for each analyte. The analytes were assayed in, at least, three batches for each HTST treatment.
The inter-assay coefficients of variation were below manufacturers' instructions for all the immune markers, and the lower limit of quantification (LLOQ) for every analyte in human milk were: 0.21 μg/L for IgA, 0.78 μg/L for IgM, 2.19 μg/L for IgG, 1.57 ng/L for TGF-β₂, 0.03 ng/L for EGF, 23.10 ng/L for adiponectin, 7.40 ng/L for ghrelin, and 11.45 ng/L for leptin.