Murine multiplex cytokine kit

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.

Right lungs were collected, weighed and immediately snap-frozen in liquid nitrogen. Samples were stored at -80⁰ C until they were processed for protein quantification and cytokine analysis as previously described [28 (link)]. Briefly, frozen lungs were homogenized in cold Tissue Protein Extraction Reagent (T-PER, Thermo Scientific) and protease inhibitor (HALT Protease Inhibitor Cocktail, Thermo Scientific) (1 mL T-PER + 10 μl HALT Protease Inhibitor Cocktail used for every 100 μg of frozen lung tissue), then centrifuged at 9000 g for 10 min at 4⁰ C. The supernatant was collected and total protein was quantified by bicinchoninic acid assay (BCA assay, Thermo Scientific Pierce) per manufacturer’s instructions. The remaining supernatant was stored at −80 °C for cytokine analysis. IFNγ concentrations in lung homogenate and first wash samples were analyzed by a murine multiplex cytokine kit (Bio-Rad, Hercules, CA). Homogenized lung samples were diluted to a total protein concentration of 500 μg/ml using a 1:1 mixture of T-PER and sample diluent (provided by manufacturer). Samples of first wash were added directly to the plate without dilution. The plates were read using a Luminex® 200™ Total System machine (Luminex Corp, Austin, Tx); data was analyzed using the LDS1.7 Software.

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.