Magnetic multiplex bead immunoassay

The concentrations for 22 plasma cytokines representing five broad categories of function (Table S1) were determined simultaneously in duplicate using a commercially available magnetic multiplex bead immunoassay (R&D Systems). Samples were processed according to the manufacturer’s instructions. Briefly, 50 μl of plasma were incubated with beads bound to a cytokine capture antibody. The 22 bead populations vary by fluorescence intensity so that they may be resolved for individual analysis. Bead cytokine concentrations were then washed and incubated with a fluorescent secondary antibody, specific for each cytokine, but fluorescing along a single channel distinct from the bead populations. The assay was performed in a 96 well plate, and analysis was performed using a Luminex Magpix instrument (Luminex, Inc., Austin, Texas). Samples from a single crewmember were processed in a single batch.

Cytokines were measured in culture supernatant from unchallenged vaginal and rectal macaque explants after 24 hours of culture. A magnetic multiplex bead immunoassay (R&D Systems, Minneapolis, MN) was used to detect MCP‐1, MIP‐1β, RANTES, IP‐10, EGF, GM‐CSF, IFN‐γ, IL‐1β, IL‐1Ra, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐15, IL‐17 and VEGF‐A on a Luminex 200 System (Bio‐Rad, Hercules, CA). Cytokine levels were normalized against total protein content as measured by a BCA protein assay (Bio‐Rad, Hercules, CA).