Luminex 100 system

Concentrations of cytokines released to apical and basolateral cell culture supernatants upon stimulation with fish PVs or LMC were measured 24 h after apical cell stimulation. Quantification of the cytokines was performed using the xMAP Technology reagents and the Luminex 100 System supported by the xPONENT software, according to the protocols recommended by the manufacturer (Merck Millipore, Massachusetts, USA). IL-6, IL-8 and CCL2 were measured using the HCYTOMAG-60K kit. IL-25 and IL-33 were measured by the HTH17MAG-14K kit, and TSLP by the HCYP2MAG-62K kit (all three kits from Merck Millipore). Stimulation of the cells with TNFα (50 ng/mL) and IL-1β (20 ng/mL) was used as a positive control for release of proinflammatory cytokines (Ozaki et al., 1996 (link); Tanaka et al., 2014 ; Tobe et al., 2002 (link)). Cytokines were measured in supernatants derived from 3 independent experiments performed in duplicates. The selection of the cytokines was made based on the published literature on the importance of these cytokines in epithelial cell damage response and allergic sensitization (Chow et al., 2010 (link); Kosaka et al., 2011 (link); Lee et al., 2015 (link); Yi et al., 2017 (link)).

CSF concentrations of 38 human cytokines/chemokines (sCD40L, EGF, Eotaxin, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC, MIP-1α, MIP-1β, TGF-α, TNFα, TNF-β, and VEGF) were simultaneously measured using a magnetic bead-based multiplex kit (HCYTMAG-60K-PX38, EMD Millipore, Billerica, MA) as per the manufacturer’s instruction. Samples were incubated with beads at 4 °C for 16 h with shaking at 500 rpm, and subsequently washed twice using a hand-held magnetic plate washer (eBioscience, San Diego, CA). The beads were incubated with the biotinylated detection antibody for 1 h at room temperature with shaking at 500 rpm, followed by incubation with streptavidin-PE for 30 min at room temperature with shaking at 500 rpm. After washing twice, the beads were resuspended in shield buffer and read on a Luminex-100 system (EMD Millipore, Billerica, MA) with a setting of 40 beads per bead set and 150 s per well. The standards (3.2–10,000 pg/mL) provided in the HCYTMAG-60 K-PX38 kit were run on each plate in duplicate, and used to calculate the concentrations of cytokines/chemokines using the Bio-Plex Manager Software (Bio-Rad, Hercules, CA).