ELISAs and multiplexed Luminex® kits were used to analyze the inflammatory biomarker profiles of MOUTH tissue. PGE2 ELISA kits were run on media collected from MOUTH tissue devices. Apical media was diluted 1:18 and basal media was diluted 1:7 for the assay. During the analysis, any samples that had undetectable levels of PGE2 or levels below the ELISA kit’s limit of detection (41.4 pg/mL) were set to the limit of detection value. All PGE2 data presented in this report is of bottom channel secreted PGE2 levels. In some cases, PGE2 remained undetectable for the 48 h sampling window. To explore cytokine and chemokine biomarker profiles of MOUTH tissue, custom Human Magnetic Luminex® Performance Assay kits (R&D Systems) containing a pre-mix of 18–20 analytes of interest were used to examine secreted soluble factors present in non-diluted basal media collected from devices. Luminex® kits used to process media collected from experiments stimulated with inflammatory cytokines IL-1β + TNF-α included analytes: MCP-1, MIP-3α, Fractalkine, GROα, IP-10, G-CSF, GM-CSF, IL-4, IL-6, IL-8, IL-10, IL-17a, IL-33, PDGF-aa, PDGF-ab/bb, RANTES, VEGF, and IFN-γ. During the analyses, any samples that had undetectable levels of a specific analyte or levels below the Luminex kit’s limit of detection for a given analyte were set to that analyte’s lower limit of detection value, whereas any sample that had levels of a specific analyte, that exceeded the Luminex kit’s limit of detection for a given analyte were set to that analytes’ upper limit of detection value. The latter occurred for IL-8, and should be considered when reviewing IL-8 secretion profiles. Undiluted media samples collected from devices were run according to the manufacturer’s protocol and analyzed using Luminex FLEXMAP 3D. The data collected were used to generate standard curves for each analytes using a 4- or 5-parameter logistic curve fit to determine the concentration of each analyte in the sample.
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