Multiplexed cytokine ELISA
Supernatants from infected cells were evaluated for 34 cytokines/chemokines by multiplex ELISA for the following analytes: CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL7/MCP-3, CCL8/MCP-2, CCL11/Eotaxin, CCL13/MCP-4, CCL20/MIP-3α, CXCL1/GROα, CXCL2/GROβ, CXCL5/ENA-78, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CXCL16, IL-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-7, IL-10, IL-12p70, IL-15, IL-16, IL-17A, IL-22, GM-CSF, MMP-9, S100A8, TNFα, TGFβ, and Trappin-2/Elafin. All antibodies and cytokine standards were purchased as antibody pairs from R&D Systems (Minneapolis, MN) or Peprotech (Rocky Hill, NJ). Individual magnetic Luminex bead sets (Luminex Corp, CA) were coupled to cytokine-specific capture antibodies according to the manufacturer’s recommendations. Samples were analyzed on a Luminex MAGPIX platform and quantified using a standard curve. For each bead region, > 50 beads were collected per analyte. The median fluorescence intensity of these beads was recorded for each bead and was used for analysis using a custom R script and a 5P regression algorithm.