Protein 200 array reader

CD4 and CD8 T cells from antibody treated myeloma-bearing mice were cultured in media alone or in the presence of 5T33 wild type or 5T33-CIITA tumor cells. Culture supernatants were harvested after 48 hours and stored at −80°C. Thawed supernatants were then analyzed using a murine multiplex cytokine kit (Bio-Rad, Hercules, CA) to detect IL-2, IL-4, IL-5, IL-10, IL-12p70, granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), and IFN-γ. Cytokines were quantified using a Bio-Plex protein 200 array reader, and data was automatically processed and analyzed by the Bio-Plex Manager Software 4.1 using standard curves generated from recombinant cytokine standards. All samples were assayed in duplicate.

Flow sorted PD-1⁺ or PD-1⁻ T cells from 5T33 myeloma-bearing mice were activated with plate-bound anti-CD3 mAb (clone 145-2C11, BD Biosciences; 5 μg/mL). Culture supernatants were harvested after 48 h and stored at −80 °C. Thawed supernatants were then analyzed using a murine multiplex cytokine kit (Bio-Rad, Hercules, CA) to detect IL-2, IL-4, IL-5, IL-10, IL-12p70, granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), and IFN-γ. Cytokines were quantified using a Bio-Plex protein 200 array reader, and data was automatically processed and analyzed using Bio-Plex Manager Software 4.1. Standard curves were generated from recombinant cytokine standards. All samples were assayed in duplicate.