V plex human chemokine panel 1

Secreted chemokines and proinflammatory cytokines were quantified from the basolateral samples of both low and high MOI infections using multiplex ELISAs (MesoScale Diagnostics): V-PLEX Human Chemokine Panel 1 (Eotaxin, Eotaxin-3, IP-10 (CXCL10), MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, TARC, IL-8) and Human ProInflammatory Tissue Culture 9-Plex (GM-CSF, IFN-γ, IL-1β, IL-10, IL-12p70, IL-2, IL-6, IL-8, TNF-α), respectively, following manufacturer instructions. Samples were diluted 1:2 for the proinflammatory cytokine panel and 1:4 for the chemokine panel in the provided reagent diluents and run in duplicate. Plates were washed as described above. All incubations were performed sealed at room temperature with shaking (3000 rpm). Before reading, 2x read buffer was added and the plates were incubated for 5 minutes (ProInflammatory 9-Plex) or read immediately (Chemokine Panel 1 V-Plex) on a Meso Scale Discovery SECTOR Imager 2400. Protein concentrations were calculated using the MesoScale Diagnostics Discovery Workbench (version 4.0.12).

Secreted interferons, cytokines, and chemokines were quantified from the apical and basolateral samples from the 48 and 96 hour post infection samples from the hNEC multistep virus growth curves. Measurements were performed using the V-PLEX Human Chemokine Panel 1 (CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26, CXCL10, and IL-8), V-PLEX Human IL-6 kit (Meso Scale Discovery), and the DIY Human IFN Lambda 1/2/3 (IL-29/28A/28B) ELISA (PBL Assay Science) according to manufacturers’ instructions. Of the 12 analytes tested, 10 (CCL2, CCL3, CCL4, CCL11, CCL17, CCL22, CXCL10, IL-8, IL-6, and IFN-λ) were specifically induced in virus-infected cultures. The amount secreted was adjusted by sample volume (either 100 or 150 μl for the apical washes, 500 μl for the basolateral supernatants) to show total pg secreted.