Ex vivo OT-1 cross-priming assay and secreted cytokine measurement

Dear Daniela,

Thanks so much for your interest in the ex vivo OT-1 cross-priming assay. In this experiment, we isolated the CD11b⁻CD11c⁺ DCs (mostly cDC1), CD11b⁺CD11c⁺ DCs, or CD11b⁺CD11c⁻ F4/80⁺ TAMs from the irradiated MC38-OVA tumors by cell sorting (BD FACSAria III/Jazz) 5 days after 8 Gy or sham irradiation ± anti-SIRPα and anti–PD-1 (fig. S19). Splenic OT-1 cells were harvested from 6- to 12-week-old C57BL/6-Tg(TcraTcrb)1100Mjb/J mice (#003831) using the CD8α+ T Cell Isolation Kit. The unstimulated OT-1 cells were cocultured with different APC subsets (OT-1:APC = 140,000:70,000) or anti-SIRPα (20  μg/ml) and/or anti–PD-1 (12.5 μg/ml) antibodies alone in RPMI 1640 with 10% FBS and 1% penicillin-streptomycin plus 50 μM β-mercaptoethanol for 48 hours in a 96-well plate. Cells were treated with GolgiPlug 6 hours before intracellular cytokine staining, and the levels of PD-1, CD44, ICOS, Granzyme B, Ki-67, and IFN-γ in OT1 cells were analyzed using flow cytometry. Secreted cytokines in the supernatant were measured using the CBA Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences #560485). The protocol of the cytometric bead array can be found on the BD website (https://www.bdbiosciences.com/en-au/products/reagents/immunoassay-reagents/cba/cba-kits/mouse-th1-th2-th17-cba-kit.560485).

Best regards,

Rodney