Multiplex Cytokine Assay Protocol

This assay was performed by the Human Immune Monitoring Center at Stanford University as previously described (47 (link)). Mouse 48 plex Procarta kit (Thermo-Fisher/Life Technologies) was used according to the manufacturer's instructions but with some modifications. The samples were added to a plate containing beads that were linked with antibodies and incubated overnight at 4°C with shaking. Then, a biotinylated detection antibody was added for 60 min at room temperature with shaking. After washing the plate, streptavidin-PE was added for 30 min at room temperature, and the plate was washed again. Reading buffer was added to the wells, and each sample was measured in duplicate. The plates were read using a Luminex 200 or a FM3D FlexMap instrument with a lower bound of 50 beads per sample per cytokine. Custom Assay Chex control beads were added to all wells.