Milliplex human cytokine chemokine growth factor panel a

Viruses in supernatant samples are inactivated with 0.5% Triton X-100 for 1 hour at room temperature. The levels of immune mediators, including cytokines, chemokines, and growth factors were quantified using the Milliplex Human Cytokine/Chemokine/Growth Factor Panel A (Merck Millipore, USA) following the manufacturer’s instructions. This system allowed quantitative measurements for 25 biomarkers: FGF-2, G-CSF, GM-CSF, IFN-α2, IFN- γ, IL-1 β, IL-1RA, IL- 2, IL-3, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 p70, IL-17A, IL-22, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-AA, PDGF-BB, TNF-α, and VEGF-A. The analysis was performed on a Luminex MAGPIX (Luminex Multiplexing Instrument, Merck Millipore) with a minimum of 50 beads collected per analyte per well. Belysa software was used to generate standard curves for each analyte using five parametric logistic fit models. Statistical data analysis was performed by using the one-way ANOVA Kruskal Wallis test with Dunn’s multiple comparison test and figures were generated using GraphPad Prism 8.0 (GraphPad Software Inc, USA).

For the cytokine release assay, 2.5x 10⁵ NK-92 cells were co-cultured or not with 0.25x10⁵ cancer cells at 37°C for 5 hours. Supernatants were harvested and IL-15, IFN-γ and TNFα were measured by multiplex assay using the MILLIPLEX MAP Human CD8+ T-Cell magnetic Bead Panel kit and MILLIPLEX Human Cytokine/Chemokine/Growth factor Panel A (Merck-Millipore). The MAGPIX ® System (Luminex Corporation) was used for data analysis according to manufacturer’s specifications. Cytokine concentrations were quantified using the Milliplex® Analyst software.

Multiplex immunoassay using macrophage culture supernatants was performed using a Luminex bead-based multiplex ELISA kit (ProcartaPlex Mouse Cytokine & Chemokine Panel 1; 26plex, Invitrogen or MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A, Millipore for MDM) according to the manufacturer’s instructions. Samples were fixed with 4% formaldehyde and subsequently washed prior to acquisition. Sample data were acquired on a MAGPIX instrument running xPONENT 4.3 software (Luminex Corp.) and analyzed using a five-parameter logistic model with an 80–120% standard acceptance range. Data was graphed using Graphpad Prism version 4.00.

Plasma cytokine/chemokine/growth factor concentrations were measured by the Luminex bead-based MILLIPLEX assay using MILLIPLEX^® Human Cytokine/Chemokine/Growth Factor Panel A (Millipore, Billerica, MA, USA) with a FlexMAP3D (Luminex) platform. Cytokine production data were analyzed using the xPONENT software, following the manufacturer’s instructions (24 (link)). The panel simultaneously analyzed 48 multiple cytokine, chemokine, and growth factor biomarkers, including sCD40L, EGF, eotaxin, FGF-2, Flt-3, ligand, fractalkine, G-CSF, GM-CSF, GRO-alpha, IFN-alpha2, IFN-gamma, IL-1 alpha, IL-1beta, 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, MIG, MIP-1 alpha, MIP-1 beta PDGF-AA, PDGF-AB/BB, RANTES, TGF-alpha, TNF-alpha, TNF-beta, and VEGF-A.