Milliplex human cytokine chemokine growth factor panel a immunology multiplex assay

Cytokine levels in each blood sample were quantified using the bead-based MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A—Immunology Multiplex Assay (EMD Millipore Corp), which is composed of analytes for target cytokines interleukin 1α (IL-1α), IL-1β, IL‑2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-17A, interferon-gamma (IFN-γ, tumor necrosis factor-alpha (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Frozen plasma samples were thawed at room temperature and analyzed following Milliplex protocol guidelines. Cytokine concentrations were measured using a Bio-Plex 200 (Bio-Rad) and Bio-Plex Manager software (Bio-Rad). Sample volume was doubled to ensure measurable levels of cytokines, and assay output data were adjusted to reflect concentrations in plasma samples. Each multiplex assay was performed in duplicate, and cytokine levels were evaluated in 3 independent measurements.

The supernatants were analyzed for cytokines (Il-2, IL6, IL10, TNF, IFNγ) during Biocompatibility Testing using the BD™ Cytometric Bead Array Human Th1/Th2 Cytokine Kit II (CBA, BD Biosciences, Germany) on a FACScan9235 device (Becton Dickinson Immocytometry Systems, Germany). For the Stability Test of the Antigens, the MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A—Immunology Multiplex Assay, Merck, Germany was used on a MagPix device (Luminex Corporation, Austin, Texas) according to the manufacturer’s instructions. Results are provided as mean fluorescence intensity (MFI) or concentration (pg/mL) where indicated.